Journal: International Journal of Molecular Sciences

Article Title: Selective Anti-Giardial Action of Indirubin: Biochemical and Functional Evidence for Inhibition of Triosephosphate Isomerase and Aldose Reductase in Giardia lamblia

doi: 10.3390/ijms27104167

Figure Lengend Snippet: Effect of IND on the proliferation and viability of Giardia lamblia trophozoites after 72 h of incubation. Trophozoites were exposed to the compound at 0, 150, 215, and 300 µM, including a vehicle control corresponding to the highest DMSO concentration used (1%). ( A ) Total trophozoite counts per experimental condition. ( B ) Comparative analysis of trophozoite cell death determined by trypan blue exclusion under each condition. Data correspond to three independent biological replicates and are presented as the mean, with bars indicating experimental standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, with statistical significance defined as p < 0.05 (* with respect to the corresponding control).

Article Snippet: Dimethylsulfoxide (DMSO) ATCC, 4-X-5.

Techniques: Incubation, Control, Concentration Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Selective Anti-Giardial Action of Indirubin: Biochemical and Functional Evidence for Inhibition of Triosephosphate Isomerase and Aldose Reductase in Giardia lamblia

doi: 10.3390/ijms27104167

Figure Lengend Snippet: Residual activity of intracellular enzymes after correction for DMSO effects. TPI ( A ) and AR ( B ) activities were measured in G. lamblia trophozoite protein extracts following 72 h exposure to IND at 150, 215, and 300 μM at 37 °C. Residual activities obtained from vehicle-treated samples (0.5%, 0.75%, and 1% DMSO, respectively) were subtracted from IND-treated samples. Data represent the enzymatic activity attributable specifically to IND after vehicle exclusion. Data correspond to three independent biological replicates and are presented as the mean, with bars indicating experimental standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, with statistical significance defined as p < 0.05 (* with respect to the corresponding control).

Article Snippet: Dimethylsulfoxide (DMSO) ATCC, 4-X-5.

Techniques: Activity Assay, Standard Deviation, Control

Journal: International Journal of Molecular Sciences

Article Title: Selective Anti-Giardial Action of Indirubin: Biochemical and Functional Evidence for Inhibition of Triosephosphate Isomerase and Aldose Reductase in Giardia lamblia

doi: 10.3390/ijms27104167

Figure Lengend Snippet: IND-induced conformational changes assessed by ANSA extrinsic fluorescence in recombinant Giardia lamblia enzymes. ( A , B ) GlTPIr and ( C , D ) GlARr were incubated under the same experimental conditions as in the enzymatic assays to directly correlate structural alterations with enzymatic activity. GlTPIr (0.2 mg/mL) and GlARr (0.5 mg/mL) were incubated in their respective buffers in the absence or presence of vehicle (DMSO) and IND for 2 h and 24 h. Following incubation, samples were exposed to ANSA (160 μM). Fluorescence emission was recorded at λex 390 nm and λem 485 nm using a spectrofluorometer. Panels A and C present fluorescence intensity in arbitrary units (a.u.), reflecting the extent of hydrophobic surface exposure. Panels B and D show normalized fluorescence values relative to the corresponding time-matched control (set to 1). Blank signals were subtracted from all measurements.

Article Snippet: Dimethylsulfoxide (DMSO) ATCC, 4-X-5.

Techniques: Fluorescence, Recombinant, Incubation, Activity Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: Selective Anti-Giardial Action of Indirubin: Biochemical and Functional Evidence for Inhibition of Triosephosphate Isomerase and Aldose Reductase in Giardia lamblia

doi: 10.3390/ijms27104167

Figure Lengend Snippet: IND promotes carbonyl stress and AGE accumulation in Giardia lamblia trophozoites. Trophozoites were treated for 72 h at 37 °C with increasing concentrations of IND (150, 215, and 300 μM), including the corresponding DMSO vehicle controls at each concentration. After treatment, cells were lysed and processed for quantification of MGO and AGEs. ( A ) Intracellular MGO levels expressed as μM per 10 6 trophozoites. ( B ) MGO levels normalized to the untreated control (set to 1). IND induced a concentration-dependent increase in MGO, reaching ~4.4 μM per 10 6 cells at 300 μM IND, corresponding to an approximate 20.6-fold increase relative to control. ( C ) AGEs levels expressed as μg/mL. ( D ) AGEs levels normalized to the untreated control (set to 1). Data correspond to three independent biological replicates and are presented as the mean, with bars indicating experimental standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, with statistical significance defined as p < 0.05 (* with respect to the corresponding control).

Article Snippet: Dimethylsulfoxide (DMSO) ATCC, 4-X-5.

Techniques: Concentration Assay, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Selective Anti-Giardial Action of Indirubin: Biochemical and Functional Evidence for Inhibition of Triosephosphate Isomerase and Aldose Reductase in Giardia lamblia

doi: 10.3390/ijms27104167

Figure Lengend Snippet: IND enhances MGO-derived protein adduct formation in Giardia lamblia trophozoites. Trophozoites were exposed for 72 h at 37 °C to IND (150, 215, and 300 μM) or to the corresponding DMSO vehicle controls (0.5%, 0.75%, and 1%, respectively, reflecting the final volume required for each IND concentration). Total protein extracts (200 μg per lane) were resolved by SDS–PAGE and transferred to PVDF membranes (0.2 μm pore size) using a semi-dry transfer system (20 V, 1 h). Membranes were incubated overnight with anti-MGO primary antibody (1:1000), followed by incubation with secondary antibody (1:2000). Immunoreactive bands were detected by enhanced chemiluminescence (luminol-based substrate) and documented using a Chemi-Doc RSC imaging system. The upper panel shows representative Western blotting detection of MGO-protein adducts in control, vehicle-treated, and IND-treated trophozoites. The lower panel presents normalized densitometric analysis, with the basal signal of the untreated control set to 1. Western blotting assays were performed. Data correspond to three independent biological replicates and are presented as the mean, with bars indicating experimental standard deviation. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test, with statistical significance defined as p < 0.05 (* with respect to the corresponding control).

Article Snippet: Dimethylsulfoxide (DMSO) ATCC, 4-X-5.

Techniques: Derivative Assay, Concentration Assay, SDS Page, Pore Size, Incubation, Imaging, Western Blot, Control, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Selective Anti-Giardial Action of Indirubin: Biochemical and Functional Evidence for Inhibition of Triosephosphate Isomerase and Aldose Reductase in Giardia lamblia

doi: 10.3390/ijms27104167

Figure Lengend Snippet: Flow cytometric analysis of apoptosis-like and necrosis in Giardia lamblia trophozoites following IND treatment. Trophozoites were incubated for 72 h under the indicated conditions and stained with Annexin V-FITC and PI to discriminate viable and dying cell populations. A total of 30,000 events per sample were analyzed. Upper panels: Controls used to validate gating and quadrant discrimination. ( 1 ) Untreated trophozoites; ( 2 ) Annexin V-FITC staining only; ( 3 ) PI staining only; ( 4 ) dual Annexin V/PI staining control; ( 5 ) apoptosis control, cells treated with H 2 O 2 (20 µM, 16 h) + Annexin V staining; ( 6 ) necrosis control, cells treated with H 2 O 2 (320 µM, 16 h) stained with Annexin V/PI staining. Vehicle controls: ( 7 – 9 ) Trophozoites treated with DMSO at 0.5%, 0.75%, and 1%, corresponding to the final concentrations required for 150, 215, and 300 µM IND, respectively. Quadrant distribution: viable cells (Ann − /PI − ; Q3), early apoptosis-like (Ann + /PI − ; Q4), late apoptosis-like (Ann + /PI + ; Q2), and necrosis (Ann − /PI + ; Q1). Untreated trophozoites maintained high viability with minimal apoptotic or necrotic populations. DMSO-treated cells exhibited a moderate, concentration-dependent increase in apoptosis-like, consistent with vehicle-induced cellular stress. Data are representative of two independent biological experiments.

Article Snippet: Dimethylsulfoxide (DMSO) ATCC, 4-X-5.

Techniques: Incubation, Staining, Control, Concentration Assay