Mrigya Babuta | Beth Israel Deaconess Medical Center (original) (raw)

Papers by Mrigya Babuta

Frontiers in cell and developmental biology, Feb 14, 2024

Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therape... more Background and aims: Granulocyte colony-stimulating factor (G-CSF) has been proposed as a therapeutic option for patients with ACLF, however clinical outcomes are controversial. We aimed at dissecting the role of G-CSF in an alcohol-induced murine model of ACLF. Methods: ACLF was triggered by a single alcohol binge (5 g/kg) in a bile duct ligation (BDL) liver fibrosis model. A subgroup of mice received two G-CSF (200 μg/kg) or vehicle injections prior to acute decompensation with alcohol. Liver, blood and brain tissues were assessed. Results: Alcohol binge administered to BDL-fibrotic mice resulted in features of ACLF indicated by a significant increase in liver damage and systemic inflammation compared to BDL alone. G-CSF treatment in ACLF mice induced an increase in liver regeneration and neutrophil infiltration in the liver compared to vehicle-treated ACLF mice. Moreover, liver-infiltrating neutrophils in G-CSF-treated mice exhibited an activated phenotype indicated by increased expression of CXC motif chemokine receptor 2, leukotriene B4 receptor 1, and calprotectin. In the liver, G-CSF triggered increased oxidative stress, type I interferon response, extracellular matrix remodeling and inflammasome activation. Circulating IL-1β was also increased after G-CSF treatment. In the cerebellum, G-CSF increased neutrophil infiltration and S100a8/9 expression, induced microglia proliferation and reactive astrocytes, which was accompanied by oxidative stress, and inflammasome activation compared to vehicletreated ACLF mice. Conclusion: In our novel ACLF model triggered by alcohol binge that mimics ACLF pathophysiology, neutrophil infiltration and S100a8/9 expression in the liver and brain indicate increased tissue damage, accompanied by oxidative stress and inflammasome activation after G-CSF treatment.

Journal of Structural Biology, May 1, 2019

Amoebiasis is a common parasitic infection in the developing world and is caused by the protist E... more Amoebiasis is a common parasitic infection in the developing world and is caused by the protist Entameoba histolytica. The proliferation of E. histolytica and its ability to invade epithelial tissues have been shown in several studies to be greatly decreased during oxidative stress. It is therefore not surprising that this amoeba has evolved several mechanisms to evade oxidative stress. Cysteine is thought to be one of the crucial molecules that help in redox defence, and a de novo cysteine biosynthetic pathway involving serine as one of the substrates has been partially elucidated in E. histolytica. Though most of the enzymes of this pathway in E. histolytica have been characterized, phosphoserine phosphatase (EhPSP), a key regulatory enzyme of the serine biosynthetic pathway, has not yet even been identified. In the current work, we identified and characterized EhPSP using various molecular, structural and functional approaches. The crystal structures of native and substrate-bound EhPSP were determined and showed the residues that play a crucial role in its phosphatase activity and substrate binding. Structural and biochemical studies indicated that EhPSP belongs to the histidine phosphatase superfamily. EhPSP-overexpressing amoebic cells were found to be more tolerant to oxidative stress. However, protection during oxidative stress was not seen when a functionally defective mutant was overexpressed. Our results clearly showed that E. histolytica has a functional PSP and that this protein participates in protecting the organism against oxidative stress.

Journal of Hepatology, 2023

Science

Adenosine monophosphate–activated protein kinase (AMPK) activity is stimulated to promote metabol... more Adenosine monophosphate–activated protein kinase (AMPK) activity is stimulated to promote metabolic adaptation upon energy stress. However, sustained metabolic stress may cause cell death. The mechanisms by which AMPK dictates cell death are not fully understood. We report that metabolic stress promoted receptor-interacting protein kinase 1 (RIPK1) activation mediated by TRAIL receptors, whereas AMPK inhibited RIPK1 by phosphorylation at Ser 415 to suppress energy stress–induced cell death. Inhibiting pS415-RIPK1 by Ampk deficiency or RIPK1 S415A mutation promoted RIPK1 activation. Furthermore, genetic inactivation of RIPK1 protected against ischemic injury in myeloid Ampkα1 -deficient mice. Our studies reveal that AMPK phosphorylation of RIPK1 represents a crucial metabolic checkpoint, which dictates cell fate response to metabolic stress, and highlight a previously unappreciated role for the AMPK-RIPK1 axis in integrating metabolism, cell death, and inflammation.

(A)E. histolytica trophozoites ... more (A)E. histolytica trophozoites were first incubated with cell tracker blue dye-labelled live CHO cells for different times, and then were fixed and stained for EhARPC1 (Alexa 488). Arrowhead indicate phagocytic cups, asterisks mark closure of cups before scission and star marks phagosomes. Scale Bar represents 10 μm. (B) The montage shows in vivo localization of GFP-EhARPC1 during the uptake of CHO cells, where CHO cells are stained with Cell tracker blue dye. Bar represents 10μm.</p

(A) The montage shows a time series of GFP-EhARPC1 expressing cells u... more (A) The montage shows a time series of GFP-EhARPC1 expressing cells undergoing erythrophagocytosis. Phagocytic cups are marked by arrowheads; RBCs in DIC are marked by yellow color arrowhead and phagocytosed RBCs by red asterisk. Bar represents 10μm. (B) Graph shows the intensity of GFP-EhARPC1 with respect to ROI during phagocytosis. Images of cells at different time points are shown in a box.</p

Journal of Surgical Research, 2015

(A) The montage shows a time series of motile trophozoites expressing... more (A) The montage shows a time series of motile trophozoites expressing GFP-EhARPC1. A number of pseudopods in different directions can be visualized and are marked by white arrowheads. Bar represents 10μm. (B) Time course of intensity of GFP-EhARPC1 (ROI) was determined at leading edge of amoeba. Snapshot of ROI selected is shown at two time points where GFP-EhARPC1 fluorescent intensity increases and then gradually decreases. (C) Quantitative determination of phagocytic events was carried out in 25 cells by randomly selecting them and counting the number of phagocytic cups, closed cups before scission, and phagosomes present in these cells. (D) E. histolytica trophozoites actively phagocytosing RBCs, incubated for different times and representing different stages of phagocytosis were stained for EhARPC1 (Alexa 488) and actin (TRITC phalloidin). Arrowhead indicate phagocytic cups, asterisks mark closure of cup before scission, stars mark phagosomes, and yellow arrowheads indicate RBCs in the process of phagocytosis. Bar represents 10μm.</p

(A) Co-precipitation of EhCaBP1 with GST-tagged protein (GST-EhARPC1 ... more (A) Co-precipitation of EhCaBP1 with GST-tagged protein (GST-EhARPC1 or GST-EhC2PK) was tested using Glutathione Sepharose beads. Beads were first loaded with recombinant GST-tagged indicated proteins and then incubated in presence of recombinant EhCaBP1. Beads were washed, and eluted proteins were detected using anti-EhCaBP1 antibody in western blots. (B) Co-immunoprecipitation of EhCaBP1 from whole amoebic lysate was done using agarose conjugated with either anti-EhARPC1 antibody or pre-immune serum (PB). The beads were washed and the eluted proteins were probed by western blot analysis using indicated antibodies. (C) Co-precipitation of his-tagged EhARPC1 with GST-tagged EhC2PK and GST-tagged EhAK1 was carried out in similar way as mentioned in (b), proteins were detected with anti-his antibody. EhAK1 was able to precipitate his-tagged EhARPC1 whereas neither GST EhC2PK nor GST alone was able to precipitate his-tagged EhARPC1. (D) Immunoprecipitation of EhARPC1 from whole-cell lysate of E. histolytica using CNBr-conjugated anti-EhAK1 antibody. The total input lysate was used for the presence of EhARPC1 and EhAK1 using respective antibodies. (E) Schematic diagram showing the organization of EhAK1 (KD and SH3) and EhARPC1 (N-ter and C-ter) constructs. (F) Co-precipitation of his-tagged EhAK1, KD and SH3 with GST-EhARPC1, N-ter and C-ter was carried out in a similar way as mentioned above in (b), only EhARPC1 and N-ter was able to pull SH3 domains. All SDS-PAGE was carried out using 10%-12%polyacrylamide gels unless otherwise mentioned.</p

(A) and (B) Immunoblot analysis of E. his... more (A) and (B) Immunoblot analysis of E. histolytica cells(HM-1:IMSS) expressing Tet-O-CAT (TOC) vector alone, or containing the cloned antisense EhARPC1 (AS) gene or sense EhARPC1 (S) in the presence and absence of tetracycline (30μg/ml). EhCaBP1 was used as an internal control. (C) Erythrocyte uptake assay was performed in over expressing (sense) and down regulated cell line (antisense) with vector alone as control. The assay was performed in the presence and the absence of tetracycline. The experiments were repeated independently three times. One-way ANOVA test was used for statistical comparisons. (D) Cells overexpressing either sense or antisense constructs of EhARPC1 were incubated with erythrocytes for the indicated times at 37°C. Cells were then fixed and stained for EhARPC1 (Alexa-488) and actin with TRITC-Phalloidin. Phagocytic cups are marked by arrowhead, star marks the just closed cup before scission and yellow arrowheads show attached RBCs at the site of phagocytosis (E) and (F) Quantitative analysis of phagocytic cups and phagosomes in over expressing (EhARPC1 sense), down regulating (EhARPC1 antisense) and Wild type HM1: MSS cell-lines, was carried out by randomly selecting 25 cells (in triplicates) and counting the number of phagocytic cups and phagosomes present in these cells. One-way ANOVA test was used for statistical comparisons. “One black star” p-value≤0.05, “Two black star” p-value≤0.005, “Three black star” p-value≤0.0005.</p

(A) Purified recombinant Kinase domain (KD) of EhAK1 or K85A (1μg) wa... more (A) Purified recombinant Kinase domain (KD) of EhAK1 or K85A (1μg) was incubated in the presence of γ-32P-ATP, MgCl2 and substrate EhARPC1-GST ((2μg) at 30°C for 1 h. KD showed phosphorylation of EhARPC1-GSTwhereas K85A mutant of EhAK1 exhibits no autophosphorylation and substrate phosphorylation activities. The products were analysed on SDS-PAGE and visualized in a phosphorimager. Red color star marks autophosphorylation band of EhAK1-KD and yellow color star marks substrate phosphorylation band. (B) Total cell- lysate of E. histolytica was passed through agarose conjugated with either anti-EhARPC1/ purified anti–EhARPC2 antibody or pre-immune serum. Co-Immunoprecipitation of EhARPC1 and EhARPC2 was checked using respective antibodies. (C) Imaging of EhARPC2 with respect to EhARPC1 and Actin was done during erythrophagocytosis where E. histolytica cells were incubated with RBCs for indicated times at 37°C. The cells were then fixed and immunostained with anti-EhARPC1 and anti EhARPC2 antibody followed by Pacific blue-410 and Alexa 488-labelled secondary antibodies respectively. F-actin was stained with TRITC phalloidin. Images with Pacific blue-410 labelled anti-EhARPC1 antibody were given pseudo-color to gray for efficient visualization. Arrowhead indicate phagocytic cups, asterisk closed cups before scission, star marks phagosome and yellow arrowheads indicate RBC to be phagocytosed. Scale Bar represents 10 μm. (D) Colocalization analysis and PCC (r) from 25 cells was done by using Olympus Fluoview FV1000 software. The values obtained by a pairwise analysis of EhARPC2 with EhARPC1, actin and EhAK1 from phagocytic cups are indicated. (E) Immunostaining was performed for amoebic cells containing EhAK1 antisense construct grown in presence or absence of 30μg/ml tet and were incubated with RBCs for 5min at 37°C. The cells were then fixed and immunostained with anti-EhAK1 and anti-EhARPC2 antibody as indicated and double stained with Pacific blue-410 and Alexa 488-labelled secondary antibodies respectively. F-actin was stained with TRITC-phalloidin. Images with Pacific blue-410 labelled anti-EhAK1 (in presence of tetracycline) is pseudo-color to gray for efficient visualization. Yellow color arrowheads show the site of RBC attachment. Scale Bar represents 10 μm.</p

(A)E. histolytica cells were in... more (A)E. histolytica cells were incubated with RBC for 5 min at 37°C. The cells were then fixed and immunostained with anti-EhARPC1 antibody followed by Pacific blue-410. F-actin was stained with TRITC phalloidin and other indicated proteins were immunostained with respective antibodies, followed by Alexa 488-labelled secondary antibody. Arrowhead indicate phagocytic cups, asterisk mark just closed cups before scission and star denotes phagosomes. Scale Bar represents 10 μm. (B) Colocalization analysis from 10 cells was done by using Olympus Fluoview FV1000 software. The Pearson’s coefficient (r) values of EhARPC1 with EhAK1, EhCaBP1, EhCaBP3 and EhC2PK from phagocytic cups are indicated. (C) Schematic representation of the different stages of phagocytosis and the localization of molecules (described in A) during these stages is summarized.</p

Frontiers in cell and developmental biology, Feb 14, 2024

Journal of Structural Biology, May 1, 2019

Journal of Hepatology, 2023

Science

Journal of Surgical Research, 2015