Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A , B ) Megakaryocytes isolated from mice bone marrow, and infected with DENV2 (MOI ~ 1) for 24 h with and without 7D. Culture supernatant was used for measuring ( A ) CXCL4 levels using ELISA, and ( B ) cell pallet was used for detecting viral genome using qRT PCR, n = 3 independent experiments for both ( P values: A : 0.0002; 0.04, B : 0.0002; 0.008). ( C , D ) Similar experiment was performed in monocytes (as CXCL4 non-producing cells) isolated from whole blood of these mice. ( C ) CXCL4 and ( D ) DENV2 genome were detected, n = 3 independent experiments for both assays ( P value: 0.0002). A similar experiment was performed in ( E , F ) human MEG-01 cell line (CXCL4 producing cells), n = 3 independent experiments ( P values: E : 0.0008; 0.01, F : 0.006) and ( G , H ) U937-DC-SIGN cell line (CXCL4 non-producing cells), n = 3 independent experiments for above assays. One-way ANOVA and Bonferroni’s post-test were used for data analysis. Data information: ( A – E , G ) One-way ANOVA and Bonferroni’s post-test were used for data analysis. Data are mean ± SEM. ( F , H ) Student’s t-test was used. Data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Isolation, Infection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A ) Sirtuin-1 (Sirt1) expression in Sirt1 −/− and WT mouse embryonic fibroblasts (MEFs) in western blot assay. Densitometry analysis independent experiments n = 3, Student’s t test was used ( P value: 0.005). Above cells were infected with DENV2 and treated with 7D as mentioned for U937-DC-SIGN in Fig. . After experiment, ( B ) WT and ( C ) Sirt1 −/− cells were used for western blot analysis for NS1, Ac-P53 and P-IRF3. ( D – I ) Densitometry data from, n = 3 independent experiments, one-way ANOVA and Bonferroni’s post-test were used ( P values: D : 0.04; 0.007; 0.01, E : 0.001, F : 0.009; 0.0002; 0.009, G : 0.005; 0.0008; 0.0006, I : 0.006; 0.002; 0.007). ( J , K ) Viral genome was quantified in WT and Sirt1 −/− MEFs pellet using qRT-PCR, n = 3 independent experiments, one-way ANOVA and Bonferroni’s post-test were used ( P values: J : 0.0013; 0.0007; 0.0005; 0.006, K : 0.004; 0.0004; 0.002; 0.003). ( L , M ) IFNα level was measured using ELISA from cell supernatant of above experiments, n = 3 independent experiments, one-way ANOVA and Bonferroni’s post-test were used ( P values: L : 0.003; 0.01; 0.0001; 0.0001, M : 0.0001; 0.0002; 0.0001; 0.0001). Data information: ( A , D – M ) Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Expressing, Western Blot, Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A ) Schematic representation of mice experiment. AG129 mice were intraperitonially (i.p.) infected with 10 5 FFU mouse-adapted DENV2 virus (P8- P23085 INDI-60) or incomplete L15 media as mock. 7D (8 mg/kg body weight) was administered (i.p.) till 4 days post-infection (DPI) and other half were injected with vehicle (PBS + 10%Tween 80) and euthanized at 6 DPI for following assays. ( B ) In a similar experiment, mice were observed till 15 DPI to obtain survival curves using the Kaplan–Meier method. The curve comparison analysis was performed between the DENV2 ( n = 12) and DENV2 + 7D ( n = 14). Log-rank (Mantle Cox) test was used for statistical analysis ( P value: 0.03). ( C ) Change in the body weight was recorded till 15 DPI, n = 7 vehicle, n = 8 mice per group, two-way ANOVA was used for data analysis, ( P values: 0.009; 0.0001; 0.0001). ( D ) Plasma CXCL4 levels from above mice was measured using ELISA at 6 DPI, n = 5 vehicle, n = 8 mice per group, one-way ANOVA and Kruskal–Wallis test were used, ( P value: 0.005). ( E , F ) DENV2 genome was quantified by qRT-PCR in ( E ) liver and ( F ) spleen tissues, respectively, n = 6 vehicle, n = 8 mice per group, one-way ANOVA and Bonferroni’s post-test were used, ( P values: E : 0.001; 0.004, F : 0.001; 0.005). ( G – M ) ( G ) Platelet, ( H ) monocytes, ( I ) neutrophil and ( J ) platelet-leukocyte aggregates were measured from peripheral blood of mice from above experiment using flow cytometry, n = 5 vehicle, n = 8 mice per group, one-way ANOVA and ( G , H : Kruskal–Wallis test) and ( I , J : Bonferroni’s post-test) were used, ( P values: G : 0.0005; 0.03, H : 0.003; 0.03, J : 0.0001; 0.005). Gating strategy is mentioned in Appendix Fig. . Plasma levels of ( K ) IL6, ( L ) TNFα and ( M ) IL1β were measured using CBA assay, n = 3 vehicle, n = 6 mice per group, Mann–Whitney U test was used, ( P values: K : 0.02; L : 0.03; M : 0.004). ( N , O ) Vascular leakage in mice blood vessels. ( N ) Image of Evan’s blue dye extravasation from the veins in abdominal region of DENV2-infected mice. ( O ) Quantification of Evan’s blue dye in different tissues, n = 5 vehicle, n = 5 for DENV2 and, n = 6 for DENV2 + 7D, two-way ANOVA was used, ( P values: 0.02; 0.03; 0.04). ( P – R ) Plasma ( P ) IFNα, ( Q ) IFNβ, n = 4 vehicle, n = 8 mice per group, and ( R ) IFNγ levels, n = 5 vehicle, n = 6 mice per group, were measured using ELISA, one-way ANOVA and Bonferroni’s post-test were used, ( P values: P : 0.006; 0.006; Q : 0.01; R : 0.03). Data information: ( C – J , O – R ) Data are mean ± SEM, and ( K – M ) median ± IQR, * P < 0.05, ** P < 0.01, *** P < 0.001, ns non-significant. .

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Infection, Virus, Injection, Comparison, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Flow Cytometry, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: ( A – D ) DENV2-specific antibodies ( A ) IgM at 3 DPI, and ( B ) IgG at 6 DPI were measured in mice plasma from the above experiments (Fig. ) using ELISA. A : n = 3 vehicle, n = 5 mice per group, one-way ANOVA and Bonferroni’s post-test, B : n = 4 vehicle, n = 8 DENV2, n = 9 DENV2 + 7D, Mann–Whitney U test was used, ( P values: A : 0.001; 0.03; B : 0.01). ( C ) FRNT 50 curves of neutralization activity of mice serum on DENV2 propagation in Vero cells, n = 3 vehicle, n = 10 mice per group. ( D ) Graph of the above values, n = 10 mice per group, Mann–Whitney U test was used ( P value: 0.02). Sera dilution assay is mentioned in Appendix Fig. . ( E ) Increased plasma cells (CD138 +ve B220 lo/-ve CD19 +ve ) percentage in 7D-treated mice spleen was measured using flow cytometry, n = 6 vehicle, n = 9 mice group, Kruskal–Wallis test is used ( P value: 0.02). ( F , G ) Spleenocytes from the above mice were used to quantitate DENV2-specific antibody secreting cells using ELISPOT assay, ( F ) representative image and ( G ) graph of the above values, n = 5 vehicle, n = 10 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: 0.0001; 0.04). ( H – J ) Intracellular levels of ( H , I ) Acetylated (Ac)-STAT3 and ( H , J ) phosphorylated ( P )-STAT3 in CD138 +ve cells was assessed by flow cytometry, n = 6 vehicle, n = 8 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: H : 0.0009; I : 0.001; 0.005; J : 0.001; 0.001). Gating strategy is mentioned in Appendix Fig. . ( K – M ) Western blot analysis of ( K , L ) Ac-STAT3 and ( K , M ) P-STAT3 from the spleenocytes normalized to β-actin. Densitometry of the above blots, n = 6 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: L : 0.001; M : 0.001). ( N , O ) Germinal center in spleen. ( N ) Immunofluorescent images of GL7-expressing follicles in spleen sections, GL7 (red) and DAPI (blue) staining. ( O ) Increased GL7-expressing follicles in 7D-treated mice, n = 8 mice per group, one-way ANOVA and Bonferroni’s post-test were used ( P values: 0.0001; 0.0002). Data information: ( A – C , E , G – J , L , M , O ) Data are mean ± SEM, and ( D ) median ± IQR, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns non-significant. Data from similar experiment in WT mice is described in Fig. . .

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Neutralization, Activity Assay, Dilution Assay, Flow Cytometry, Enzyme-linked Immunospot, Western Blot, Expressing, Staining

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: DENV2 infection experiment was performed in bone marrow derived macrophages (BMDMs), isolated from AG129 mice, in presence of 7D. A blocking antibody to IFNλ2/3 (5 µg/ml, standardization of working concentration is mentioned in Fig. EV4L below) was used to investigate the effects of type-III IFN. ( A ) Viral genome was quantified in cell pellets using qRT-PCR, n = 3 independent experiments ( P values: 0.0011; 0.0002; 0.03; 0.0014). ( B – E ) Relative gene expressions of interferon-stimulated genes (ISGs), ( B ) Oas1a, ( C ) Isg15, ( D ) Trim69, and ( E ) Mx1 were measured from above experiments, n = 3 independent experiments, ( P values: B : 0.0001; 0.0013; 0.0001; 0.0001, C : 0.0001; 0.0001; 0.0012; 0.01; 0.0001, D : 0.0006; 0.01; 0.0008; 0.0001, E : 0.0001; 0.0001; 0.0014; 0.009; 0.0001). ( F ) IFN λ2/3 levels were measured in the supernatant of these cells using ELISA, n = 3 independent experiments, ( P values: 0.004; 0.02; 0.002). ( G , H ) Relative gene expressions of ifnl2 and ifnl3 were quantified from the above cell pellets, n = 3 independent experiments, ( P values: G : 0.001; 0.001; 0.008, H : 0.001; 0.003; 0.001). ( I – K ) Western blot analysis for P-p38:p38 and P-IRF3:IRF3. ( J , K ) Densitometry data from, n = 3 independent experiments, ( P values: J : 0.007; 0.003; 0.001, K : 0.0006; 0.0002; 0.0003). ( L ) Concentration-dependent effect of blocking antibody against type-III IFN was tested on viral replication. DENV2 mRNA was quantified from pellets of DENV2-infected U937-DC-SIGN cells in presence of increasing concentration (1, 5 and 10 µg/ml) of blocking antibody against IFN-λ2/3 Ab, n = 3 independent experiments, ( P values: 0.001; 0.002; 0.0002). One-way ANOVA and Bonferroni’s post-test were used for above analysis. Data information: ( A – H , J – L ) One-way ANOVA and Bonferroni’s post-test were used. Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Infection, Derivative Assay, Isolation, Blocking Assay, Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: As mentioned in the above Fig. , a similar experiment was performed in CXCR3 −/− and WT C57BL/6 mice at 6 DPI. ( A , B ) Spleen was isolated and processed for measuring dengue viral genome copies were quantified in ( A ) CXCR3 −/− , n = 5 vehicle, n = 7 mice per group, one-way ANOVA and Kruskal–Wallis test, ( P values: 0.0002; 0.03), and ( B ) WT, n = 6 vehicle, n = 7 mice per group, one-way ANOVA and Bonferroni’s post-test, ( P values: 0.004; 0.04) using qRT-PCR. ( C , D ) IFNα levels were measured in plasma of these mice (C) CXCR3 −/− , n = 5 vehicle, n = 7 mice per group, and ( D ) WT, n = 6 vehicle, n = 9 mice per group, one-way ANOVA and Bonferroni’s post-test were used for both cases, ( P values: C : 0.0003, D : 0.01). ( E , F ) DENV2-specific IgG levels were measured in mice plasma of ( E ) CXCR3 −/− , n = 5 vehicle, n = 7 mice per group, and ( F ) WT, n = 5 vehicle, n = 6 mice per group, using ELISA. One-way ANOVA and Bonferroni’s post-test were used for both cases, ( P values: E : 0.003; 0.0013, F : 0.001). ( G – J ) FRNT 50 curves and graph of neutralization activity of mice plasma was measured in ( G , H ) CXCR3 −/− , n = 3 vehicle, n = 7 mice per group, and ( I , J ) WT, n = 6 vehicle, n = 7 mice per group. Mann–Whitney U test was used for analysis for both cases, ( P value: 0.003). Sera dilution assay is mentioned in Appendix Fig. . Data information: ( A – F , G , I ) Data are mean ± SEM, and ( H , J ) median ± IQR, * P < 0.05, ** P < 0.01, ns non-significant. .

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Isolation, Quantitative RT-PCR, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay, MANN-WHITNEY, Dilution Assay

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: As mentioned in the Fig. , a similar experiment was performed in AG129 mice treated with 7D and STAT3-inhibitor Stattic (10 mg/kg/body weight, referred concentration from vendor’s manual). ( A , B ) DENV2 viral RNA was quantified in liver and spleen respectively using qRT-PCR, n = 8 mice per group, ( P values: A : 0.0001; 0.0001; 0.003, B : 0.0001; 0.0003; 0.04). ( C , D ) Anti-dengue IgG was measured from the serum of mice using ELISA, n = 8, ( P values: 0.0001; 0.0005; 0.003). ( E – G ) Ac-STAT3 and P-STAT3 were measured in spleenocytes by western blot and normalized with total STAT3. Densitometry of the above blots, n = 4 vehicle, n = 6 mice per group ( P values: F : 0.0008; 0.0001, G : 0.01). One-way ANOVA and Bonferroni’s post-test were used for all above analysis. Data information: ( A – D , F , G ) One-way ANOVA and Bonferroni’s post-test were used. Data are mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns=non-significant.

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Concentration Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: EMBO Molecular Medicine

Article Title: 7D, a small molecule inhibits dengue infection by increasing interferons and neutralizing-antibodies via CXCL4:CXCR3:p38:IRF3 and Sirt1:STAT3 axes respectively

doi: 10.1038/s44321-024-00137-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Human PF4 ELISA (Elabscience, USA).

Techniques: Isolation, Sequencing, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, Lysis, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction