scholarly journals A novel approach for in vivo measurement of mouse red cell redox status

Blood ◽  
2011 ◽  
Vol 118 (13) ◽  
pp. 3694-3697 ◽  
Author(s):  
Xiuling Xu ◽  
Katharina von Löhneysen ◽  
Katrin Soldau ◽  
Deborah Noack ◽  
Andrew Vu ◽  
...  
Keyword(s):  
Redox Balance ◽  
Redox Status ◽  
In Vivo Measurement ◽  
Novel Approach ◽  
Physiologic Function ◽  
Versatile Tool ◽  
Thiol Redox ◽  
Cell Redox Status

Abstract Maintenance of a reducing redox balance is a critical physiologic function of red cells (RBC) that can be perturbed in variety of RBC pathologies. Here we describe a new approach to evaluate in vivo RBC redox status using a redox sensitive GFP (roGFP2) sensor under control of a β-globin mini-promoter, directing expression specifically to erythroid cells. RoGFP2 expressing RBCs demonstrate ratiometric and reversible shifts in fluorescence on exposure to oxidants and reductants. We demonstrate that roGFP2 expressing RBC can be used to monitor thiol redox status during in vitro phenylhydrazine treatment and over the course of in vivo RBC aging, where a shift to a more oxidized state is observed in older cells. Thus, roGFP2 transgenic mice are a new and versatile tool that can be used to probe how RBC redox status responds in the context of drug therapy, physiologic stressors and pathologic states.

A Novel Approach for In Vivo Measurement of Red Cell Redox Status

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2036-2036 ◽  
Author(s):  
Xiuling Xu ◽  
Katharina von Loehneysen ◽  
Deborah Noack ◽  
Andrew Vu ◽  
Jeff S. Friedman
Keyword(s):  
Fluorescence Emission ◽  
Redox Status ◽  
Red Cells ◽  
Cysteine Residues ◽  
Excitation Wavelength ◽  
Red Cell ◽  
Reducing Conditions ◽  
Cell Redox Status

Abstract Abstract 2036 Maintenance of a reducing redox balance is a critical physiologic function of red cell metabolic machinery. Perturbation of this balance, whether inherited or acquired, is found in a variety of red cell pathologies. Methods for evaluation of red cell redox status include direct approaches such as determining glutathione (GSH, GSSG) levels, and indirect approaches such as measuring fluorescence of oxidation sensitive dyes. Here we describe an alternative method for evaluation of red cell redox status that can be used in vivo and in real-time assays. Engineered variants of GFP possessing two solvent accessible cysteine residues function as molecular redox sensors with distinct fluorescence characteristics. Excitation spectrum shifts upon the oxidation of cysteine residues forming a disulfide. A higher ratio of fluorescence when comparing excitation at 405nm versus 488nm indicates rising levels of oxidized GFP and a shift in cellular redox status. To validate redox GFPs in erythroid cells, we first performed in vitro assays with MEL cells over-expressing several related GFP sensors (ro-GFPs), selecting the brightest molecule (roGFP2) for further study. The sensor function of roGFP2 in MEL cells was verified by stimulation with exogenous oxidant (1mM H202) or reductant (10 mM DTT) as shown in the figure below. In order to create a physiologic in vivo model for study of red cell redox status, transgenic mice expressing roGFP2 specifically in the erythroid lineage were generated. roGFP2 expressing red cells demonstrate the expected shift in fluorescence upon exposure to H202 or t-butyl peroxide in a short-term assay. In vivo, we have measured red cell lifespan (using biotin-labeling) in roGFP2 transgenic animals to follow redox status of red cells as a function of cell age. Expression of roGFP2 has no effect on red cell survival. Interestingly, when comparing old red cells (age > 50days) with younger cells (age < 50days), a shift in GFP fluorescence ratio indicating that a higher fraction of the sensor is oxidized in the aged cells was observed. This observation is consistent with the hypothesis that metabolic changes, in particular a decline in ability to reduce oxidative damage, contribute to red cell senescence. We are generating several murine strains with defined red cell defects also expressing roGFP2 in order to assess the role of changes in intra-erythrocyte redox status in a range of pathologic conditions. In vitro and in vivo assays using roGFP2 transgenic cells/mice are in process to determine the potential utility of this system as a screen for hematoxicity of drugs and other compounds. Figure 1 Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Figure 1. Evaluation of roGFP2 function in MEL cells. The Y-axis shows fluorescence emission as a function of excitation wavelength (X-axis)—showing a shift when cells are exposed to oxidizing or reducing conditions. Disclosures: No relevant conflicts of interest to declare.

scholarly journals TXNRD3 supports male fertility via the redox control of spermatogenesis

2021 ◽  
Author(s):  
Qianhui Dou ◽  
Anton A Turanov ◽  
Marco Mariotti ◽  
Jae Yeon Hwang ◽  
Huafeng Wang ◽  
...  
Keyword(s):  
Critical Role ◽  
Thioredoxin Reductase ◽  
Redox Status ◽  
Fertilization Rate ◽  
Male Reproduction ◽  
Redox Control ◽  
Thiol Redox ◽  
Knockout Animals

Thioredoxin/glutathione reductase (TGR, TXNRD3) is a thiol oxidoreductase of unknown function composed of thioredoxin reductase and glutaredoxin domains. This NADPH-dependent enzyme evolved by gene duplication within the Txnrd family, is expressed in the testes and can reduce both thioredoxin and glutathione in vitro. To characterize the function of TXNRD3 in vivo, we generated a strain of mice with the deletion of Txnrd3 gene. We show that Txnrd3 knockout mice are viable and without discernable gross phenotypes, but TXNRD3 deficiency leads to fertility impairment in male mice. Txnrd3 knockout animals exhibit a lower fertilization rate in vitro, a sperm movement phenotype and an altered redox status of thiols. Proteomic analyses revealed a broad range of substrates reduced by TXNRD3 during sperm maturation, presumably as a part of quality control. The results show that TXNRD3 plays a critical role in male reproduction via the thiol redox control of spermatogenesis.

scholarly journals HSP90-dependent PUS7 overexpression facilitates the metastasis of colorectal cancer cells by regulating LASP1 abundance

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Dan Song ◽  
Ming Guo ◽  
Shuai Xu ◽  
Xiaotian Song ◽  
Bin Bai ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Intellectual Development ◽  
Molecular Mechanisms ◽  
Hsp90 Inhibitor ◽  
Pseudouridine Synthase ◽  
Novel Approach ◽  
Cell Metastasis ◽  
Underlying Mechanisms

Abstract Background Pseudouridine synthase (PUS) 7 is a member of the PUS family that catalyses pseudouridine formation. It has been shown to be involved in intellectual development and haematological malignancies. Nevertheless, the role and the underlying molecular mechanisms of PUS7 in solid tumours, such as colorectal cancer (CRC), remain unexplored. This study elucidated, for the first time, the role of PUS7 in CRC cell metastasis and the underlying mechanisms. Methods We conducted immunohistochemistry, qPCR, and western blotting to quantify the expression of PUS7 in CRC tissues as well as cell lines. Besides, diverse in vivo and in vitro functional tests were employed to establish the function of PUS7 in CRC. RNA-seq and proteome profiling analysis were also applied to identify the targets of PUS7. PUS7-interacting proteins were further uncovered using immunoprecipitation and mass spectrometry. Results Overexpression of PUS7 was observed in CRC tissues and was linked to advanced clinical stages and shorter overall survival. PUS7 silencing effectively repressed CRC cell metastasis, while its upregulation promoted metastasis, independently of the PUS7 catalytic activity. LASP1 was identified as a downstream effector of PUS7. Forced LASP1 expression abolished the metastasis suppression triggered by PUS7 silencing. Furthermore, HSP90 was identified as a client protein of PUS7, associated with the increased PUS7 abundance in CRC. NMS-E973, a specific HSP90 inhibitor, also showed higher anti-metastatic activity when combined with PUS7 repression. Importantly, in line with these results, in human CRC tissues, the expression of PUS7 was positively linked to the expression of HSP90 and LASP1, and patients co-expressing HSP90/PUS7/LASP1 showed a worse prognosis. Conclusions The HSP90-dependent PUS7 upregulation promotes CRC cell metastasis via the regulation of LASP1. Thus, targeting the HSP90/PUS7/LASP1 axis may be a novel approach for the treatment of CRC.

scholarly journals Plasma cells shape the mesenchymal identity of ovarian cancers through transfer of exosome-derived microRNAs

2021 ◽  
Vol 7 (9) ◽  
pp. eabb0737
Author(s):  
Zhengnan Yang ◽  
Wei Wang ◽  
Linjie Zhao ◽  
Xin Wang ◽  
Ryan C. Gimple ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Ovarian Cancer Cells ◽  
Mesenchymal Phenotype ◽  
Ovarian Cancers ◽  
Novel Approach

Ovarian cancer represents a highly lethal disease that poses a substantial burden for females, with four main molecular subtypes carrying distinct clinical outcomes. Here, we demonstrated that plasma cells, a subset of antibody-producing B cells, were enriched in the mesenchymal subtype of high-grade serous ovarian cancers (HGSCs). Plasma cell abundance correlated with the density of mesenchymal cells in clinical specimens of HGSCs. Coculture of nonmesenchymal ovarian cancer cells and plasma cells induced a mesenchymal phenotype of tumor cells in vitro and in vivo. Phenotypic switch was mediated by the transfer of plasma cell–derived exosomes containing miR-330-3p into nonmesenchymal ovarian cancer cells. Exosome-derived miR-330-3p increased expression of junctional adhesion molecule B in a noncanonical fashion. Depletion of plasma cells by bortezomib reversed the mesenchymal characteristics of ovarian cancer and inhibited in vivo tumor growth. Collectively, our work suggests targeting plasma cells may be a novel approach for ovarian cancer therapy.

scholarly journals THER-05. GENETICALLY STABLE POLIOVIRUS VECTOR CARRYING H3.3K27M ANTIGEN FOR TREATMENT OF DIFFUSE MIDLINE GLIOMA BY INTRAMUSCULAR INJECTION

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii472-iii472
Author(s):  
Mubeen Mosaheb ◽  
Daniel Landi ◽  
Elena Dobrikova ◽  
Michael Brown ◽  
Yuanfan Yang ◽  
...  
Keyword(s):  
T Cell ◽  
Antigen Presentation ◽  
Intramuscular Injection ◽  
Cd8 T Cell ◽  
Cytokine Profiles ◽  
Cell Immunity ◽  
Novel Approach ◽  
Diffuse Midline Glioma

Abstract BACKGROUND H3 K27M-mutant diffuse midline glioma (DMG) is invariably lethal. Viruses naturally engage innate immunity, induce antigen presentation, and mediate CD8 T cell priming against foreign antigens. Polioviruses, in particular, are uniquely tropic for dendritic cells (DC) and potently activate DC, inducing Th1-dominant cytokine profiles, CD8 T cell immunity, and enhanced epitope presentation. Thus, poliovirus is ideally suited for vectored delivery of signature tumor neoantigens, e.g. the H3 K27M feature of DMG. However, poliovirus vector design is inherently limited by genetic instability and the underlying neuropathogenicity of poliovirus. METHODS We created a genetically stable, polio:rhinovirus chimera vector devoid of neuropathogenicity and modified for stable expression of the HLA-A2 restricted H3.3 K27M antigen (RIPO (H3.3)). RESULTS RIPO(H3.3) infects, activates, and induces H3.3K27M antigen presentation in DCs in vitro. Given intramuscularly in vivo, RIPO(H3.3) recruits and activates DCs with Th1-dominant cytokine profiles, efficiently primes H3.3K27M-specific CD8 T cells, induces antigen-specific CD8 T cell migration to the tumor site, delays tumor growth, and enhances survival in murine tumor models. CONCLUSION This novel approach leverages the unique ability of polioviruses to activate DCs while simultaneously introducing the H3.3 K27M antigen. In this way, DCs are activated optimally in situ, while being simultaneously infected to express/present tumor antigen. RIPO(H3.3), given by intramuscular injection, will be evaluated in a clinical trial for children with H3 K27M-mutant diffuse midline glioma.

scholarly journals The Role of Herbal Bioactive Components in Mitochondria Function and Cancer Therapy

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Fangfang Tao ◽  
Yanrong Zhang ◽  
Zhiqian Zhang
Keyword(s):  
Cancer Therapy ◽  
Cancer Cell ◽  
Apoptotic Cell ◽  
Redox Status ◽  
Cancer Therapies ◽  
Bioactive Components ◽  
Atp Production

Mitochondria are highly dynamic double-membrane organelles which play a well-recognized role in ATP production, calcium homeostasis, oxidation-reduction (redox) status, apoptotic cell death, and inflammation. Dysfunction of mitochondria has long been observed in a number of human diseases, including cancer. Targeting mitochondria metabolism in tumors as a cancer therapeutic strategy has attracted much attention for researchers in recent years due to the essential role of mitochondria in cancer cell growth, apoptosis, and progression. On the other hand, a series of studies have indicated that traditional medicinal herbs, including traditional Chinese medicines (TCM), exert their potential anticancer effects as an effective adjunct treatment for alleviating the systemic side effects of conventional cancer therapies, for reducing the risk of recurrence and cancer mortality and for improving the quality of patients’ life. An amazing feature of these structurally diverse bioactive components is that majority of them target mitochondria to provoke cancer cell-specific death program. The aim of this review is to summarize the in vitro and in vivo studies about the role of these herbs, especially their bioactive compounds in the modulation of the disturbed mitochondrial function for cancer therapy.

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