Construction of a robust polarity sensitive platform and its application for tracking of lipid droplets decrease under oxidative stress in live cells

Oxidative imbalance plays an essential role in the progression of many diseases that include cancer and neurodegenerative diseases. Aromatic amino acids (AAA) such as phenylalanine and tryptophan have the capability of escalating oxidative stress because of their involvement in the production of Reactive Oxygen Species (ROS). Here, we use D2O (heavy water) probed stimulated Raman scattering microscopy (DO-SRS) and two Photon Excitation Fluorescence (2PEF) microscopy as a multimodal imaging approach to visualize metabolic changes in HeLa cells under excess AAA such as phenylalanine or trytophan in culture media. The cellular spatial distribution of de novo lipogenesis, new protein synthesis, NADH, Flavin, unsaturated lipids, and saturated lipids were all imaged and quantified in this experiment. Our studies reveal ∼10% increase in de novo lipogenesis and the ratio of NADH to flavin, and ∼50% increase of the ratio of unsaturated lipids to saturated lipid in cells treated with excess phenylalanine or trytophan. In contrast, these cells exhibited a decrease in the protein synthesis rate by ∼10% under these AAA treatments. The cellular metabolic activities of these biomolecules are indicators of elevated oxidative stress and mitochondrial dysfunction. Furthermore, 3D reconstruction images of lipid droplets were acquired and quantified to observe their spatial distribution around cells’ nuceli under different AAA culture media. We observed a higher number of lipid droplets in excess AAA conditions. Our study showcases that DO-SRS imaging can be used to quantitatively study how excess AAA regulates metabolic activities of cells with subcellular resolution in situ.

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Effect of Probiotic and Amla Powder Supplementation on Metabolic Profile and Milk Production of Summer Stressed Buffaloes Residing in Heavy Metal Polluted Areas of Ludhiana

Indian Journal of Animal Research ◽

10.18805/ijar.b-4270 ◽

2021 ◽

Author(s):

Pranesh V. Yeotikar ◽

Shashi Nayyar ◽

Chanchal Singh ◽

C.S. Mukhopadhyay ◽

Sandeep Sodhi Kakkar ◽

...

Keyword(s):

Oxidative Stress ◽

Heavy Metal ◽

Milk Production ◽

Metabolic Profile ◽

Antioxidant Status ◽

Live Cells ◽

Metal Exposure ◽

Heavy Metal Exposure ◽

Exposed Area ◽

Oral Supplementation

Background: Oxidative stress is a unified concept for the assessment of metabolic status of buffaloes affected by a cocktail of heavy metal exposure from environment and it may significantly affect the metabolic profile and milk production during summer season. The present study aimed at evaluation of effect of probiotic (Saccharomyces cerevisiae strain 1026) and amla powder (Embilica officinalis) supplementation on levels of heavy metals, antioxidant status, oxidative stress in lymphocytes, metabolic profile and milk production of summer stressed buffaloes exposed to environmental heavy metal pollution. Methods: Twenty summer stressed lactating Murrah buffaloes of the same age group, close parity and in early lactation from the heavy metal exposed area were divided into two groups: Control (without supplementation) and Treatment group (supplemented with Saccharomyces cerevisiae1026) @ 50 billion live cells / animal /day and Amla powder @ 86 mg/kg body weight for 30 days. Blood and milk samples were collected from both the groups on the day of starting of supplementation (i.e. day 0) and on there after days 15 and 30 and analyzed for antioxidant status, metabolic profile, milk yield and quality.Result: Oral supplementation of probiotic and amla powder for 30 days improved the antioxidant status, metabolic profile and milk production of summer stressed buffaloes residing in heavy metal exposed area. The percentage monetary gains from buffaloes fed probiotic and amla powder was 13.68% more as compared to control.

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Aurone Derivative Revealing the Metabolism of Lipid Droplets and Monitoring Oxidative Stress in Living Cells

Analytical Chemistry ◽

10.1021/acs.analchem.0c00456 ◽

2020 ◽

Vol 92 (9) ◽

pp. 6631-6636 ◽

Cited By ~ 3

Author(s):

Kangnan Wang ◽

Shuyue Ma ◽

Yanyan Ma ◽

Yuping Zhao ◽

Miaomiao Xing ◽

...

Keyword(s):

Oxidative Stress ◽

Lipid Droplets ◽

Living Cells

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Lipid droplets alleviate cadmium induced cytotoxicity in Saccharomyces cerevisiae

Toxicology Research ◽

10.1039/c6tx00187d ◽

2017 ◽

Vol 6 (1) ◽

pp. 30-41 ◽

Cited By ~ 5

Author(s):

Selvaraj Rajakumar ◽

Vasanthi Nachiappan

Keyword(s):

Oxidative Stress ◽

Saccharomyces Cerevisiae ◽

Reactive Oxygen Species ◽

Lipid Accumulation ◽

Lipid Droplets ◽

Reactive Oxygen ◽

Oxygen Species

Cadmium (Cd) induces oxidative stress that generates reactive oxygen species (ROS) and increased lipid accumulation.

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Multiplex coherent anti-Stokes Raman scattering microspectroscopy detection of lipid droplets in cancer cells expressing TrkB

Scientific Reports ◽

10.1038/s41598-020-74021-z ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Tiffany Guerenne-Del Ben ◽

Vincent Couderc ◽

Ludovic Duponchel ◽

Vincent Sol ◽

Philippe Leproux ◽

...

Keyword(s):

Cancer Cells ◽

Lipid Droplets ◽

Live Cells ◽

Label Free ◽

Colorectal Cancer Cell Lines ◽

Invasive Method ◽

Dye Staining ◽

Cytoplasmic Lipid Droplets ◽

Anti Stokes ◽

Cell Fixation

Abstract For many years, scientists have been looking for specific biomarkers associated with cancer cells for diagnosis purposes. These biomarkers mainly consist of proteins located at the cell surface (e.g. the TrkB receptor) whose activation is associated with specific metabolic modifications. Identification of these metabolic changes usually requires cell fixation and specific dye staining. MCARS microspectroscopy is a label-free, non-toxic, and minimally invasive method allowing to perform analyses of live cells and tissues. We used this method to follow the formation of lipid droplets in three colorectal cancer cell lines expressing TrkB. MCARS images of cells generated from signal integration of CH2 stretching modes allow to discriminate between lipid accumulation in the endoplasmic reticulum and the formation of cytoplasmic lipid droplets. We found that the number of the latter was related to the TrkB expression level. This result was confirmed thanks to the creation of a HEK cell line which over-expresses TrkB. We demonstrated that BDNF-induced TrkB activation leads to the formation of cytoplasmic lipid droplets, which can be abolished by K252a, an inhibitor of TrkB. So, MCARS microspectroscopy proved useful in characterizing cancer cells displaying an aberrant lipid metabolism.

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Polarity sensitive probes for super resolution STED microscopy

10.1101/107334 ◽

2017 ◽

Author(s):

E Sezgin ◽

F Schneider ◽

V Zilles ◽

E Garcia ◽

D Waithe ◽

...

Keyword(s):

Plasma Membrane ◽

Lipid Membrane ◽

Membrane Lipids ◽

Super Resolution ◽

Membrane Vesicles ◽

Molecular Organization ◽

Live Cells ◽

Sted Microscopy ◽

Two Photon Microscopy ◽

Polarity Sensitive

AbstractThe lateral organization of molecules in the cellular plasma membrane plays an important role in cellular signaling. A critical parameter for membrane molecular organization is how the membrane lipids are packed (or ordered). Polarity sensitive dyes are powerful tools to characterize such lipid membrane order, employing for example confocal and two-photon microscopy. The investigation of potential lipid nanodomains, however, requires the use of super resolution microscopy. Here, we test the performance of the polarity sensitive membrane dyes Di-4-ANEPPDHQ, Di-4-AN(F)EPPTEA and NR12S in super resolution STED microscopy. Measurements on cell-derived membrane vesicles, in the plasma membrane of live cells, and on single virus particles show the high potential of these dyes for probing nanoscale membrane heterogeneity.

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269IN VITRO FERTILITY OF BOAR SPERMATOZOA PRESERVED AT 10^°C FOR 22 DAYS

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab269 ◽

2004 ◽

Vol 16 (2) ◽

pp. 255

Author(s):

H. Funahashi

Keyword(s):

Oxidative Stress ◽

Functional Status ◽

Pregnancy Rate ◽

Seminal Plasma ◽

Artificial Insemination ◽

Fluorescence Assay ◽

Live Cells ◽

Sperm Viability ◽

Boar Spermatozoa

Fertility of boar spermatozoa as determined following artificial insemination seems to be maintained during liquid preservation at 10–15°C for several days, although prolonged liquid preservations reduce the pregnancy rate rapidly. However, it is not clear if spermatozoa can penetrate into oocytes in an IVF system even after a prolonged liquid preservation. Oxidative stress could also be one of the possible detrimental factors in liquid preservation of spermatozoa. In the present study, fertility of liquid-preserved spermatozoa was examined using an IVM-IVF system. Whether cysteine can improve the fertility was also determined. Spermatozoa (from four Berkshires) was resuspended at 1×108 cells mL−1 in Modena solution containing 15% (v/v) boar seminal plasma and 0 or 5mM cysteine after washing 3 times. Sperm suspensions (1mL) were then preserved at 10°C for 22 days following a program for cooling down (to 15°C for 4h, keeping at 15°C for 12h and then to 10°C for 6h). At Days 1, 8, 15 and 22 after the start of preservation, spermatozoa (5×105 cells mL−1) were co-cultured with IVM oocytes in an IVM/IVF system (Funahashi et al., 1997 Biol Reprod 57, 49–53). Viability and functional status of spermatozoa were also examined at Days 8 and 15 of preservation by using LIVE/DEAD sperm viability kit and CTC fluorescence assay. Data (mean±SEM) from 4–6 replicates were analyzed by ANOVA and Fisher’s protected LSD test. When spermatozoa that had been preserved without cysteine (Cys−) were used, penetration rates were not different (P>0.05) from those with cysteine (Cys+) at Day 8 of preservation (91.4±3.4% in Cys− and 99.3±0.7% in Cys+), but lower (P<0.02) at Days 15 and 22 (72.6±13.6% and 33.8±8.4% in Cys−; 94.8±2.1% and 71.1±10.8% in Cys+, respectively). Both viability and proportion of uncapacitated live cells were higher (P<0.05) in Cys+ than Cys− at Days 8 and 15. These results demonstrate that boar spermatozoa can penetrate into oocytes in vitro even after a liquid preservation at 10°C for 22 days and that cysteine can improve the viability and penetrability in vitro of spermatozoa during liquid preservation. Supported by the Ito Foundation.

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Localization of dichlorofluorescin in cardiac myocytes: implications for assessment of oxidative stress

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.3.h982 ◽

2000 ◽

Vol 278 (3) ◽

pp. H982-H990 ◽

Cited By ~ 60

Author(s):

Luther M. Swift ◽

Narine Sarvazyan

Keyword(s):

Oxidative Stress ◽

Cardiac Muscle ◽

Live Cells ◽

Cardiac Mitochondria ◽

Mitochondrial Marker ◽

Cardiac Muscle Cells ◽

Preferential Localization ◽

Carbonyl Cyanide ◽

Mitochondrial Distribution ◽

Mitotracker Red

Localization and staining features of the oxidant-sensitive fluorescent probe 2′7′-dichlorofluorescin (DCFH) were evaluated in isolated cardiac muscle cells. Cardiomyocytes rapidly accumulated the probe and retained steady levels of DCFH and its highly fluorescent oxidized product dichlorofluorescein (DCF) in probe-free medium for 1.5 h. DCF was associated with mitochondria and was released by the proton ionophore carbonyl cyanide m-chlorophenylhydrazone but not by saponin, which permeabilizes the plasma membrane. A mitochondrial distribution of DCF was also suggested by experiments with the mitochondrial marker MitoTracker Red, in which quenching was observed between DCF and MitoTracker Red in live cells. Isolated cardiac mitochondria rapidly accumulated DCF, and high micromolar concentrations of the probe inhibited ADP-stimulated respiration rate. The study provides an information base essential for the interpretation and design of experiments with DCF as a marker of oxidative stress in cardiac muscle and reveals preferential localization of the probe in mitochondria.

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Development of the quadripolar meiotic apparatus in Funaria spore mother cells: analysis by means of anti-microtubule drug treatments

Journal of Cell Science ◽

10.1242/jcs.93.2.267 ◽

1989 ◽

Vol 93 (2) ◽

pp. 267-277

Author(s):

C. H. BUSBY ◽

B. E. GUNNING

Keyword(s):

Electron Microscopy ◽

Lipid Droplets ◽

Immunofluorescence Microscopy ◽

Video Recording ◽

Live Cells ◽

Spindle Poles ◽

Microtubule Organizing Centres ◽

Drug Treatments ◽

Mother Cells ◽

Dependent Processes

Microtubule-dependent processes in Funaria hygrometrica spore mother cells (SMCs) were analysed by monitoring the effects of colchicine and oryzalin on pre-meiotic and meiotic events. The techniques used were electron microscopy, immunofluorescence microscopy of microtubules (MTs)and continuous video recording of events in treated and recovering live cells sampled at various stages of sporogenesis. Inferences drawn from previous work that the SMC plastids serve as MT-organizing centres were confirmed in so far as MT recovery in MT-depleted cells starts at the tips of the plastids. The MTs that emanate from these regions are required for positioning the plastids in the tetrahedral conformation, which defines the meiotic poles, for positioning lipid droplets in clusters at these poles and for positioning and holding the nucleus in the tetrahedral cage. If released, the nucleus can be moved by a non-MT system. Other phenomena not controlled by MTs are plastid elongation, maintenance of the tetrahedral conformation-when the MTs are absent (during divisions or as a result of drug treatment) and (probably) development of the organelle band that spans the cell between divisions I and II. In cells treated during division, when there is nonuclear envelope, the pattern of MT recovery is different: the plastids are inactive as microtubule-organizing centres (MTOCs) but MTs reappear among the chromosomes. Spindles capable of transporting chromosomes regenerate. However, the importance of interactions between nucleus and plastids is highlighted by cases in which treatmenthas resulted in: (1) movement of the nucleus out of the quadripolar plastid cage; and (2) loss of the MTs at plastid tips that normally contribute to the spindle poles; in such cases quadripolarity is lost even though functional spindles return. Plastid MTOC activity returns when the nuclear envelope isin place, i.e. in interkinesis and after telophase II.

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