mitotempo (mt  (Merck & Co)

Journal: Frontiers in Microbiology

Article Title: Priming Astrocytes With HIV-Induced Reactive Oxygen Species Enhances Their Trypanosoma cruzi Infection

doi: 10.3389/fmicb.2020.563320

Figure Lengend Snippet: Effect of Trypanosoma cruzi infection on astrocyte ROS level and its consequences on parasite infection and multiplication. (A) Timeline of the Tc infection of cultured human astrocytes. Parameters were measured at T1 (orange arrow, 24 h after Tc exposure). (B) mROS level was measured by flow cytometry using MitoSOX in uninfected astrocytes – Ctrl – and Tc-infected astrocytes. (C) cROS level was measured by flow cytometry using DCFDA (assumed to be proportional to the concentration of hydrogen peroxide) in uninfected astrocytes – Ctrl – and Tc-infected astrocytes. (D) Cell death levels (positive staining for annexin-V and 7-AAD) in uninfected (Ctrl) and Tc-infected astrocytes analyzed by flow cytometry. (E) Timeline of the exposure of cultured astrocytes to tert-Butyl hydroperoxide (TBH; blue arrow) followed by Tc infection (green arrow, 1 h after TBH-exposure). Parameters were measured at T3 (orange arrow, 48 h after Tc exposure). (F) Cell death levels by flow cytometry (positive staining for annexin-V and 7-AAD) in unexposed (Ctrl) and TBH-exposed cells (at increasing concentrations as indicated), in non-infected (black columns) and Tc-infected (gray columns) astrocytes. (G) Tc-infection rate analysis in unexposed (Ctrl) and TBH-exposed cells (at increasing concentrations as indicated). Three representative dot plots representation are shown. For each diagram, a square depicted the percentage of Tc-infected cells (GFP-positive). (H) Tc-multiplication quantification by flow cytometry as mean fluorescence intensity (MFI, expressed in arbitrary units) in unexposed (Ctrl) and TBH-exposed cells (at increasing concentrations as indicated). (I) Timeline of the exposure of cultured astrocytes to ROS scavengers (MT: MitoTEMPO; AA: ascorbic acid; blue arrow) followed by Tc infection (green arrow, 18 h after scavengers exposure). Parameters were measured at T3 (orange arrow, 48 h after Tc exposure). (J) Tc-infection rate analysis in non-scavenged and scavenged (with MT, or AA) T. cruzi -infected cells. (K) Tc-multiplication quantification by flow cytometry as MFI in non-scavenged and scavenged (with MT, or AA) T. cruzi -infected cells. (L) Timeline of the exposure of cultured astrocytes to pro-oxidant condition (TBH, 100 μM during 1 h), ROS scavenging (AA: ascorbic acid), and Tc infection (green arrow, 18 h after AA exposure). Parameters were measured at T3 (orange arrow, 48 h after Tc exposure). (M) Astrocyte ROS level (% cell DCFDA+) and Tc infection rate (% cell Tc+) analysis in control and TBH-treated astrocytes, with and without scavenging (AA). Graphics are showing values obtained from three independent experiments. Data are given as the mean ± SD. NS (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Production of cellular and mitochondrial ROS was evaluated using DCFDA and MitoSOX assays, respectively as follows: Also, astrocytes were pre-treated for 18 h with the antioxidant ascorbic acid (AA) or with MitoTEMPO® (MT; Sigma-Aldrich), a well-known mitochondria-specific superoxide scavenger, to validate the in vitro model and to demonstrate ROS functionality.

Techniques: Infection, Cell Culture, Flow Cytometry, Concentration Assay, Staining, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Priming Astrocytes With HIV-Induced Reactive Oxygen Species Enhances Their Trypanosoma cruzi Infection

doi: 10.3389/fmicb.2020.563320

Figure Lengend Snippet: Effect of trypomastigotes-derived extracellular vesicles (EVs) in astrocytes ROS concentration and Trypanosoma cruzi infection. (A) Scanning electron microscopy (SEM) showing clusters of trypomastigotes-derived EVs formed by ultracentrifugation. The bar size is indicated. (B) Biochemical characterization of EVs purified from trypomastigotes by western blot to verify the presence of SAPA (shed acute-phase antigen) and Tc-TASV-C antigens and the absence of SR62 ( T. cruzi cytoplasmic antigen). (C) Biochemical characterization of EVs purified from astrocytes. The presence of CD63, CD9, and absence of calnexin was determined by western blot from astrocyte-derived EVs. (D) Timeline of the EVs exposure (blue arrow) in cultured astrocytes followed by Tc infection (green arrow, 24 h after EVs exposure). Parameters were measured at T3 (orange arrow, 48 h after Tc exposure). (E) Tc-infection rate measured as GFP+ cells analysis in EVs non-exposed astrocytes followed by Tc infection (Tc), and EVs-exposed cells [EVs from normal astrocytes (EVs A), Tc-infected astrocytes (EVs A/Tc), and EVs from free trypomastigotes (EVs Tc)]. Two representative dot plots obtained by flow cytometry showing EVs non-exposed astrocytes (Ctrl, left panel) and EVs (from trypomastigotes) exposed-astrocytes (EVs-Tc, right panel). In each diagram, a square depicted the percentage of Tc-infected cells (GFP-positive). (F) Astrocyte death levels (measured as positive staining for annexin-V and 7-AAD) in EVs non-exposed astrocytes followed by Tc infection (Ctrl), and EVs-exposed cells [with EVs obtained from cell sources defined in item (E) ]. (G) Tc-infection rate measured as GFP+ cells analysis in EVs from free trypomastigotes (EVs Tc) at dilutions 1/100 (0.06 μg/well), 1/10 (0.6 μg/well), and pure (6 μ/well). (H) Cellular and mitochondrial ROS level (using DCFDA – represented in gray columns and MitoTEMPO – represented in black columns, respectively) as MFI, in astrocytes (exposed to EVs from sources described in E ). (I) Timeline of the EVs exposure (blue arrow) in cultured astrocytes simultaneously infected with Tc (green arrow). Parameters were measured at T2 (orange arrow, 48 h after Tc + EVs exposure). (J) Timeline of the cultured astrocytes infected with Tc (green arrow) and stimulated with EVs (blue arrow, 24 h after Tc exposure). Parameters were measured at T3 (orange arrow, 48 h after EVs exposure). (K) Tc-infection rate measured as GFP+ cells analysis in EVs non-exposed astrocytes but Tc-infected (Ctrl), and EVs from free trypomastigotes-exposed cells simultaneously with Tc infection. (L) Tc-infection rate measured as GFP+ cells in EVs non-exposed astrocytes but Tc-infected (Ctrl), and EVs from free trypomastigotes-exposed cells after infection with Tc. Graphics are showing values obtained from three independent experiments. Data are given as the mean ± SD Significant differences are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively.

Article Snippet: Production of cellular and mitochondrial ROS was evaluated using DCFDA and MitoSOX assays, respectively as follows: Also, astrocytes were pre-treated for 18 h with the antioxidant ascorbic acid (AA) or with MitoTEMPO® (MT; Sigma-Aldrich), a well-known mitochondria-specific superoxide scavenger, to validate the in vitro model and to demonstrate ROS functionality.

Techniques: Derivative Assay, Concentration Assay, Infection, Electron Microscopy, Purification, Western Blot, Cell Culture, Flow Cytometry, Staining

Journal: Frontiers in Microbiology

Article Title: Priming Astrocytes With HIV-Induced Reactive Oxygen Species Enhances Their Trypanosoma cruzi Infection

doi: 10.3389/fmicb.2020.563320

Figure Lengend Snippet: Role of HIV-induced ROS on Trypanosoma cruzi infection and multiplication in astrocytes. (A) Timeline of the HIV exposure (blue arrow) of cultured astrocytes followed by Tc infection (green arrow, 48 h after HIV exposure). Parameters were measured at T3 (orange arrow, 24 h after Tc exposure). (B) Cellular ROS levels (as a percentage of DCFDA-positive cells, on left y-axis) and HIV infection efficiency (measured by flow cytometry as a percentage of GFP-positive cells, on the right y-axis) using two different inoculums at T0 (+: 8 μg/ml, ++: 80 μg/ml of p24 antigen). (C) Tc-infection rate measured by flow cytometry analysis in HIV non-exposed (Tc) and HIV-exposed cells (at the two inoculums indicated as HIV+/Tc and HIV++/Tc). (D) Tc-multiplication quantification by flow cytometry as mean fluorescence intensity (MFI, expressed in arbitrary units) in HIV non-exposed (Tc) and HIV-exposed astrocytes (at the two inoculums indicated as HIV+/Tc and HIV++/Tc). (E) Timeline of the HIV exposure (blue arrow) of cultured astrocytes followed by scavenging (MT: MitoTEMPO; AA: ascorbic acid; violet arrow, 24 h after HIV exposure), and Tc infection (green arrow, 48 h after HIV exposure). Parameters were measured at T3 (orange arrow, 24 h after Tc exposure). (F) Tc-infection rate measured by flow cytometry analysis in HIV non-exposed astrocytes followed by Tc infection (Tc) and in HIV-exposed cells (at the two inoculums as indicated), then scavenged (with MT, or AA), and finally exposed to Tc. A control without pretreatment with scavengers is included (HIV + Tc). Graphics are showing values obtained from three independent experiments. Data are given as the mean ± SD Significant differences are indicated by * p < 0.05, ** p < 0.01, and *** p < 0.001, respectively.

Article Snippet: Production of cellular and mitochondrial ROS was evaluated using DCFDA and MitoSOX assays, respectively as follows: Also, astrocytes were pre-treated for 18 h with the antioxidant ascorbic acid (AA) or with MitoTEMPO® (MT; Sigma-Aldrich), a well-known mitochondria-specific superoxide scavenger, to validate the in vitro model and to demonstrate ROS functionality.

Techniques: Infection, Cell Culture, Flow Cytometry, Fluorescence

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: Effect of the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p) on metabolic parameters in rats fed a normal chow (CT) and high fat diet (HFD) fed rats.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: Protein levels of ( A ) fibronectin, ( B ) periostin, ( C ) α-smooth muscle actin (SMA) and ( D ) vimentin in heart from control rats fed a normal chow (CT) and rats fed a high fat diet (HFD) treated with vehicle or with the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p). Bars graphs represent the mean ± SEM of 6–8 animals. Protein densitometry was expressed in arbitrary units (AU) once normalized to stain-free gel for protein.* p < 0.05; ** p < 0.01 vs. CT group. † p < 0.05, †† p < 0.01 vs. HFD group.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques: Staining

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: ( A ) Representative microphotographs and ( B ) quantification of total mucin levels in colon from control animals fed a normal chow (CT) and animals fed a high fat diet (HFD) treated with vehicle or with the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p) stained with Alcian Blue (AB)/periodic acid-Schiff (PAS) examined by light microscopy (magnification 20×). Bar graphs represent the mean ± SEM of 5–6 animals normalized to for CT group. *** p < 0.001 vs. CT group; †† p < 0.01 vs. HFD group.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques: Staining, Light Microscopy

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: Alpha and beta diversity indexes in rats fed a normal chow (CT) and high fat diet (HFD) and treated with the antioxidant mitochondrial MitoTempo (MT; 0.7 mg/Kg/day i.p).

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: Boxplot showing total relative abundance of reads of the four most abundant taxa at Phylum level in the gut microbiota ( A ) Firmicutes , ( B ) Bacteroidetes , ( C ) Protobacteria and ( D ) Tenericutes in feces from control animals fed a normal chow (CT) and animals fed a high fat diet (HFD) treated with vehicle or with the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p). Upper, middle and lower lines represent first quartiles, medians and third quartiles. The whiskers represent a 1.5 * inter-quartile range. Data are expressed as percentage of total reads. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. CT group. † p < 0.05, †† p < 0.01 vs. HFD group.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: LEfSe analysis showing taxonomic differential abundance at family level in feces from control animals fed a normal chow (CT) and animals fed a high fat diet (HFD) treated with vehicle or with the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p). ( A ) Significantly different families among CT, HFD and HFD+MT groups. ( B ) Significantly enriched and depleted families between CT and HFD groups. ( C ) Significantly enriched and depleted families between HFD and HFD + MT groups. The length of the horizontal bars represents the LDA score (effect size). p < 0.05; LDA score > 3.0.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: LEfSe analysis showing taxonomic differential abundance at genus level in feces from control animals fed a normal chow (CT) and animals fed a high fat diet (HFD) treated with vehicle or with the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p). ( A ) Significantly different genera among CT, HFD and HFD+MT groups. ( B ) Significantly enriched and depleted genera between CT and HFD groups. ( C ) Significantly enriched and depleted genera between HFD and HFD + MT groups. The length of the horizontal bars represents the LDA score (effect size). p < 0.05; LDA score > 3.0.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques:

Journal: Antioxidants

Article Title: The Interaction between Mitochondrial Oxidative Stress and Gut Microbiota in the Cardiometabolic Consequences in Diet-Induced Obese Rats

doi: 10.3390/antiox9070640

Figure Lengend Snippet: Boxplot showing total relative abundance of metabolic pathways related to gut microbiota in ( A ) butanoate metabolism, ( B ) propanoate metabolism and ( C ) glutathione metabolism and ( D ) percentage of bacteria involved in lipopolysaccharyde (LPS) production in feces from control animals fed a normal chow (CT) and animals fed a high fat diet (HFD) treated with vehicle or with the mitochondrial antioxidant MitoTempo (MT; 0.7 mg/Kg/day i.p). Upper, middle and lower lines represent first quartiles, medians and third quartiles. The whiskers represent a 1.5 * inter-quartile range. Data are expressed as percentage of total reads. * p < 0.05; ** p < 0.01; *** p < 0.001 vs. CT group. † p < 0.05 vs. HFD group.

Article Snippet: The mitochondrial targeted antioxidant MitoTempo (MT) was obtained from Merck Sigma Aldrich (St. Louis, MO, USA).

Techniques: