scholarly journals Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

2021 ◽  
Vol 27 ◽  
Author(s):  
Szilvia Brunner ◽  
Dániel Varga ◽  
Renáta Bozó ◽  
Róbert Polanek ◽  
Tünde Tőkés ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Phase Distribution ◽  
Control Level ◽  
Repair Process ◽  
Quantitative Detection ◽  
Cycle Phase ◽  
Dependent Manner ◽  
Pronounced Increase ◽  
Radiation Induced ◽  
U251 Cells

The quantitative detection of radiation caused DNA double-strand breaks (DSB) by immunostained γ-H2AX foci using direct stochastic optical reconstruction microscopy (dSTORM) provides a deeper insight into the DNA repair process at nanoscale in a time-dependent manner. Glioblastoma (U251) cells were irradiated with 250 keV X-ray at 0, 2, 5, 8 Gy dose levels. Cell cycle phase distribution and apoptosis of U251 cells upon irradiation was assayed by flow cytometry. We studied the density, topology and volume of the γ-H2AX foci with 3D confocal microscopy and the dSTORM superresolution method. A pronounced increase in γ-H2AX foci and cluster density was detected by 3D confocal microscopy after 2 Gy, at 30 min postirradiation, but both returned to the control level at 24 h. Meanwhile, at 24 h a considerable amount of residual foci could be measured from 5 Gy, which returned to the normal level 48 h later. The dSTORM based γ-H2AX analysis revealed that the micron-sized γ-H2AX foci are composed of distinct smaller units with a few tens of nanometers. The density of these clusters, the epitope number and the dynamics of γ-H2AX foci loss could be analyzed. Our findings suggest a discrete level of repair enzyme capacity and the restart of the repair process for the residual DSBs, even beyond 24 h. The dSTORM superresolution technique provides a higher precision over 3D confocal microscopy to study radiation induced γ-H2AX foci and molecular rearrangements during the repair process, opening a novel perspective for radiation research.

scholarly journals Role of leukemia inhibitor factor (LIF) in decidualisation of murine uterine stromal cells in vitro

2004 ◽  
Vol 181 (3) ◽  
pp. 477-492 ◽  
Author(s):  
AA Fouladi Nashta ◽  
CV Andreu ◽  
N Nijjar ◽  
JK Heath ◽  
SJ Kimber
Keyword(s):  
In Vitro Culture ◽  
Stromal Cells ◽  
Control Level ◽  
Calf Serum ◽  
Prostaglandin E ◽  
Dependent Manner ◽  
Alp Activity ◽  
Dose Dependent ◽  
In Cells

Decidualisation of uterine stromal cells is a prerequisite for implantation of the embryo in mice. Here we have used an in vitro culture system in which stromal cells decidualise as indicated by a number of markers, including an increase in alkaline phosphatase (ALP) activity. The latter was used as a quantitative marker of decidualisation in the presence of low (2%) fetal calf serum. Prostaglandin E(2) (PGE(2)), which is known to induce decidualisation, increased ALP activity, and this effect was blocked in a dose-dependent manner by indomethacin. Leukemia inhibitory factor (LIF) was then examined, but it had no effect on PGE(2) secretion. However, LIF suppressed ALP activity in a dose-dependent manner in the presence of 2% serum, while an inhibitor of LIF that competes for binding to its receptor reversed the effect of LIF and increased ALP activity above the control level. In serum-free cultures, stromal cells differentiated rapidly, and no differences were observed between LIF-treated and untreated cultures. Stromal cells produce LIF during in vitro culture, and this peaked at 48 h. Freshly collected stromal cells from both day-2 and -4 pregnant mice expressed mRNA for the LIF receptor, and the transcript level was higher in cells isolated on day 4. However, no differences were observed in the relative levels of transcripts in cells from day 2 and day 4 after culture, nor were there differences between the LIF-treated cultures and controls. Therefore, in this study, we have shown that LIF suppresses decidualisation of murine uterine stromal cells in the presence of serum, this is not due to the regulation of PGE(2) secretion by stromal cells.

scholarly journals Placenta Derived Mesenchymal Stem Cells Hosted on RKKP Glass-Ceramic: A Tissue Engineering Strategy for Bone Regenerative Medicine Applications

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Mario Ledda ◽  
Marco Fosca ◽  
Angela De Bonis ◽  
Mariangela Curcio ◽  
Roberto Teghil ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Regenerative Medicine ◽  
Glass Ceramic ◽  
Phase Distribution ◽  
Sol Gel ◽  
Pcr Analysis ◽  
Cycle Phase ◽  
Cell Delivery ◽  
Limiting Factor

In tissue engineering protocols, the survival of transplanted stem cells is a limiting factor that could be overcome using a cell delivery matrix able to support cell proliferation and differentiation. With this aim, we studied the cell-friendly and biocompatible behavior of RKKP glass-ceramic coated Titanium (Ti) surface seeded with human amniotic mesenchymal stromal cells (hAMSCs) from placenta. The sol-gel synthesis procedure was used to prepare the RKKP glass-ceramic material, which was then deposited onto the Ti surface by Pulsed Laser Deposition method. The cell metabolic activity and proliferation rate, the cytoskeletal actin organization, and the cell cycle phase distribution in hAMSCs seeded on the RKKP coated Ti surface revealed no significant differences when compared to the cells grown on the treated plastic Petri dish. The health of of hAMSCs was also analysed studying the mRNA expressions of MSC key genes and the osteogenic commitment capability using qRT-PCR analysis which resulted in being unchanged in both substrates. In this study, the combination of the hAMSCs’ properties together with the bioactive characteristics of RKKP glass-ceramics was investigated and the results obtained indicate its possible use as a new and interesting cell delivery system for bone tissue engineering and regenerative medicine applications.

scholarly journals Vanadyl Sulfate Treatment Stimulates Proliferation and Regeneration of Beta Cells in Pancreatic Islets

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Samira Missaoui ◽  
Khémais Ben Rhouma ◽  
Mohamed-Tahar Yacoubi ◽  
Mohsen Sakly ◽  
Olfa Tebourbi
Keyword(s):  
Blood Glucose ◽  
Insulin Sensitivity ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Control Level ◽  
Diabetic Rats ◽  
Diabetic Group ◽  
Dependent Manner ◽  
Glucose Levels ◽  
Blood Glucose Levels

We examined the effects of vanadium sulfate (VOSO4) treatment at 5 and 10 mg/kg for 30 days on endocrine pancreas activity and histology in nondiabetic and STZ-induced diabetic rats. In diabetic group, blood glucose levels significantly increased while insulinemia level markedly decreased. At the end of treatment, VOSO4at a dose of 10 mg/Kg normalized blood glucose level in diabetic group, restored insulinemia, and significantly improved insulin sensitivity. VOSO4also increased in a dose-dependent manner the number of insulin immunopositive beta cells in pancreatic islets of nondiabetic rats. Furthermore, in the STZ-diabetic group, the decrease in the number of insulin immunopositive beta cells was corrected to reach the control level mainly with the higher dose of vanadium. Therefore, VOSO4treatment normalized plasma glucose and insulin levels and improved insulin sensitivity in STZ-experimental diabetes and induced beta cells proliferation and/or regeneration in normal or diabetic rats.

scholarly journals The Role of Copper in the Regulation of Ferroportin Expression in Macrophages

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2259
Author(s):  
Aneta Jończy ◽  
Rafał Mazgaj ◽  
Ewa Smuda ◽  
Beata Żelazowska ◽  
Zuzanna Kopeć ◽  
...  
Keyword(s):  
Iron Homeostasis ◽  
Control Level ◽  
Raw 264.7 ◽  
Protein Abundance ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Iron Storage ◽  
Raw 264.7 Macrophages ◽  
Transcriptional Suppression ◽  
Lps Treatment

The critical function of ferroportin (Fpn) in maintaining iron homeostasis requires complex and multilevel control of its expression. Besides iron-dependent cellular and systemic control of Fpn expression, other metals also seem to be involved in regulating the Fpn gene. Here, we found that copper loading significantly enhanced Fpn transcription in an Nrf2-dependent manner in primary bone-marrow-derived macrophages (BMDMs). However, prolonged copper loading resulted in decreased Fpn protein abundance. Moreover, CuCl2 treatment induced Fpn expression in RAW 264.7 macrophages at both the mRNA and protein level. These data suggest that cell-type-specific regulations have an impact on Fpn protein stability after copper loading. Transcriptional suppression of Fpn after lipopolysaccharide (LPS) treatment contributes to increased iron storage inside macrophages and may result in anemia of inflammation. Here, we observed that in both primary BMDMs and RAW 264.7 macrophages, LPS treatment significantly decreased Fpn mRNA levels, but concomitant CuCl2 stimulation counteracted the transcriptional suppression of Fpn and restored its expression to the control level. Overall, we show that copper loading significantly enhances Fpn transcription in macrophages, while Fpn protein abundance in response to CuCl2 treatment, depending on macrophage type and factors specific to the macrophage population, can influence Fpn regulation in response to copper loading.

scholarly journals Effect of genetic variants in FAS and let-7a on radiation-induced intestinal toxicity in the treatment of prostate cancer

2021 ◽  
Author(s):  
Honglei Guo ◽  
Feng Yuan ◽  
Yancui Zhu ◽  
Ling He
Keyword(s):  
Prostate Cancer ◽  
Dependent Manner ◽  
Intestinal Toxicity ◽  
Rectal Volume ◽  
Obvious Association ◽  
Radiation Induced ◽  
Computational Analyses ◽  
Putative Marker ◽  
Dose Dependent

IntroductionThe present study aimed to explore the effects of pri-let-7a-1 rs10739971 and FAS-670 rs1800682 polymorphisms on the pathogenesis of radiation induced intestinal toxicity in prostate cancer (PC) patients.Material and methods380 PC patients with or without signs of intestinal toxicity were enrolled to study the effects of let-7a rs10739971 and FAS-670 rs1800682 polymorphisms on rectal volume and the risk of intestinal toxicity. In addition, real-time PCR, Western-blot analysis, immunohistochemistry, luciferase assays and computational analyses were performed to explore the mechanism underlying the role of let-7a rs10739971 polymorphism in radiation induced intestinal toxicity.ResultsThe let-7a rs10739971 polymorphism but not the FAS-670 rs1800682 polymorphism was closely related to the risk of radiation induced intestinal toxicity featured by a high rectal volume. In addition, there was no obvious association between the rectal volume and the genotype and allele frequencies of FAS -670 rs1800682 and Pri-let-7a-1 rs10739971 polymorphisms. The GG genotype of let-7a rs10739971 polymorphism reduced let-7a expression but enhanced FAS expression. In addition, the intestinal toxicity (-) group showed a much higher level of let-7a and a much lower level of FAS than the intestinal toxicity (+) group. FAS was a virtual target gene of let-7a, which decreased FAS protein expression in a dose-dependent manner.ConclusionsThe GG genotype of pri-let-7a-1 rs10739971 polymorphism could increase the risk of radiation induced intestinal toxicity in PC patients. Therefore, the pri-let-7a-1 rs10739971 polymorphism could be used as a putative marker to predict the risk of intestinal toxicity in PC patients undergoing radiotherapy.

scholarly journals Honokiol ameliorates radiation-induced brain injury via the activation of SIRT3

2020 ◽  
Vol 48 (10) ◽  
pp. 030006052096399
Author(s):  
Guixiang Liao ◽  
Zhihong Zhao ◽  
Hongli Yang ◽  
Xiaming Li
Keyword(s):  
Oxidative Stress ◽  
Brain Injury ◽  
Inflammatory Responses ◽  
Tissue Injury ◽  
Interleukin 1 ◽  
Dependent Manner ◽  
Ht22 Cells ◽  
Tnf Α ◽  
Radiation Induced ◽  
Cox 2

Objective Sirtuin 3 (SIRT3) plays a vital role in regulating oxidative stress in tissue injury. The aim of this study was to evaluate the radioprotective effects of honokiol (HKL) in a zebrafish model of radiation-induced brain injury and in HT22 cells. Methods The levels of reactive oxygen species (ROS), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β) were evaluated in the zebrafish brain and HT22 cells. The expression levels of SIRT3 and cyclooxygenase-2 (COX-2) were measured using western blot assays and real-time polymerase chain reaction (RT-PCR). Results HKL treatment attenuated the levels of ROS, TNF-α, and IL-1β in both the in vivo and in vitro models of irradiation injury. Furthermore, HKL treatment increased the expression of SIRT3 and decreased the expression of COX-2. The radioprotective effects of HKL were achieved via SIRT3 activation. Conclusions HKL attenuated oxidative stress and pro-inflammatory responses in a SIRT3-dependent manner in radiation-induced brain injury.

scholarly journals A Polyphenol Rich Muscadine Grape Extract Reduces Triple Negative Breast Cancer Cell Migration in Association with Changes in Cell Morphology and Motility-Related Proteins

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 325-325
Author(s):  
Patricia Gallagher ◽  
Marianne Collard ◽  
Heather Brown-Harding ◽  
Elisabeth Tallant
Keyword(s):  
Breast Cancer ◽  
Cell Migration ◽  
Confocal Microscopy ◽  
Triple Negative Breast Cancer ◽  
Cell Shape ◽  
Molecular Mechanisms ◽  
Triple Negative ◽  
Dependent Manner ◽  
Grape Extract ◽  
Muscadine Grape

Abstract Objectives Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by the lack of estrogen receptors, progesterone receptors and over-expression of the human epidermal growth factor receptor 2, limiting targeted treatment.  TNBC disproportionally affects ethnic minorities and younger women and has a high propensity to metastasize, often within 5 years of diagnosis, making it one of the most aggressive breast cancer subtypes.  We showed that treatment with a proprietary muscadine grape extract (MGE) reduced the growth and metastasis of TNBC in mice.  Muscadine grapes (V. Rotundifolia) are rich in polyphenols and extracts produced from muscadine grape seed and skin are marketed as nutraceuticals for their anti-oxidant, anti-inflammatory, and anti-cancer properties.  The goal of these studies was to determine the molecular mechanisms for the reduction in metastatic growth by MGE. Methods A proprietary extract was prepared from muscadine grape seeds and skins.  Migration of MDA-MB-231 and BT-549 cells was measured by a scratch wound assay, cell shape was visualized by confocal microscopy and mRNA/proteins that participate in cell migration/motility were measured by RT-PCR and western blot hybridization. Results The extract reduced the migration of MDA-MB-231 and BT-549 TNBC cells in a dose-dependent manner.  The reduction in cell migration was associated with MGE-induced alterations in cell shape and actin filament organization, visualized by confocal microscopy.  The extract caused an apparent loss of cell polarization in MDA-MB-231 cells and a reduction in the presence of filopodia in BT-549 cells.  The MGE-induced reduction in migration and alterations in cell shape and polarization were associated with a decrease in Rho kinase ROCK1/2 mRNA and protein as well as both the mRNA and protein expression of RHAMM, a protein that is implicated in both cell motility and breast cancer progression. Conclusions These results demonstrate that a proprietary MGE reduces TNBC cell migration, in association with changes in cell shape and cytoskeleton as well as proteins that regulate migration and motility, suggesting that treatment of TNBC patients with MGE may slow or prevent metastatic progression. Funding Sources Chronic Disease Research Fund.

Contribution of Electron and Confocal Microscopy in the Study ofLeishmania–Macrophage Interactions

2004 ◽  
Vol 10 (5) ◽  
pp. 656-661 ◽  
Author(s):  
Birgitta Rasmusson ◽  
Albert Descoteaux
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Protozoan Parasite ◽  
Laser Scanning Microscopy ◽  
Sand Fly ◽  
Confocal Laser ◽  
Mammalian Host ◽  
Dependent Manner ◽  
Phagosome Maturation ◽  
Physical Barrier

Promastigotes of the protozoan parasite genusLeishmaniaare inoculated into a mammalian host when an infected sand fly takes a bloodmeal. Following their opsonization by complement, promastigotes are phagocytosed by macrophages. There, promastigotes differentiate into amastigotes, the form of the parasite that replicates in the phagolysosomal compartments of host macrophages. Although the mechanisms by which promastigotes survive the microbicidal consequence of phagocytosis remain, for the most part, to be elucidated, evidence indicates that glycoconjugates play a role in this process. One such glycoconjugate is lipophosphoglycan, an abundant promastigote surface glycolipid. Using quantitative electron and confocal laser scanning microscopy approaches, evidence was provided thatL. donovanipromastigotes inhibit phagolysosome biogenesis in a lipophosphoglycan-dependent manner. This inhibition correlates with an accumulation of periphagosomal F-actin, which may potentially form a physical barrier that preventsL. donovanipromastigote-containing phagosomes from interacting with endocytic vacuoles. Inhibition of phagosome maturation may constitute a strategy to provide an environment propitious to the promastigote-to-amastigote differentiation.

Coincident microvillar actin bundle disruption and perinuclear actin sequestration in anoxic proximal tubule

2000 ◽  
Vol 278 (6) ◽  
pp. F886-F893 ◽  
Author(s):  
Peter White ◽  
R. Brian Doctor ◽  
Rolf H. Dahl ◽  
Jing Chen
Keyword(s):  
Control Level ◽  
Dependent Manner ◽  
Differential Interference Contrast ◽  
Differential Interference Contrast Microscopy ◽  
Proximal Tubular Cells ◽  
Actin Bundle ◽  
Tubular Cells ◽  
Contrast Microscopy ◽  
Proximal Tubular ◽  
Interference Contrast Microscopy

The present studies investigated acute disruption of microvillar actin cytoskeleton and actin association with other cytoskeletal components in ATP-depleted rabbit proximal tubular cells. Video-enhanced differential-interference contrast microscopy and confocal microscopy were used to follow the fate of F-actin during the disruption of microvilli. Within individual cells, all microvilli collapsed simultaneously. Microvillar actin filaments underwent a parallel decrease in length. Using a sequential cytoskeletal extraction protocol and electron microscopy, we revealed in the present studies the coincident sequestration of a distinct, perinuclear pool of actin that was primarily absent in control cells. Actin sequestration progressed in a duration-dependent manner, occurring as early as 15 min of anoxia when cellular ATP dropped to <5% of control level. Phalloidin staining and depolymerization treatment showed the majority (>90%) of this sequestered actin to be F-actin. A microvillar actin bundling protein villin was also sequestered in the same perinuclear complex of anoxic proximal tubules. In conclusion, the present results demonstrate a coincident microvillar actin bundle disruption and the perinuclear sequestration of F-actin in ATP-depleted proximal tubular cells.

Regulation of KCa-channel activity by cyclic ADP-ribose and ADP-ribose in coronary arterial smooth muscle

1998 ◽  
Vol 275 (3) ◽  
pp. H1002-H1010 ◽  
Author(s):  
Pin-Lan Li ◽  
Ai-Ping Zou ◽  
William B. Campbell
Keyword(s):  
Smooth Muscle ◽  
Time Course ◽  
Control Level ◽  
Internal Surface ◽  
Threshold Concentration ◽  
Dependent Manner ◽  
Channel Activity ◽  
Arterial Smooth Muscle ◽  
Concentration Dependent Manner ◽  
Cyclic Adp Ribose

The enzymatic pathway responsible for the production and metabolism of cyclic ADP-ribose (cADP-R) in small bovine coronary arteries was characterized, and the role of cADP-R and ADP-ribose (ADP-R) in the regulation of the activity of large-conductance Ca2+-activated K+(KCa) channels was determined in vascular smooth muscle cells (SMC) prepared from these vessels. We found that cADP-R and ADP-R were produced when the coronary arterial homogenates were incubated with 1 mM β-NAD. The time course of the enzyme reactions showed that the maximal conversion rate (1.37 ± 0.03 nmol ⋅ min−1 ⋅ mg protein−1) of β-NAD to cADP-R was reached after 3 min of incubation. As incubation time was prolonged, the production of ADP-R was increased to a maximal rate of 3.66 ± 0.03 nmol ⋅ min−1 ⋅ mg protein−1, whereas cADP-R production decreased. Incubation of the homogenate with cADP-R produced a time-dependent increase in the synthesis of ADP-R. Comparison of coronary arterial microsomes with cytosols shows that the production of both cADP-R and ADP-R in microsomes was significantly greater. In excised inside-out membrane patches of single coronary SMC, the KCa channels were activated when β-NAD, the precursor for both cADP-R and ADP-R, was applied to the internal surface. This effect of β-NAD may be associated with the production of ADP-R, because the KCa-channel activity was increased by ADP-R in a concentration-dependent manner. The open-state probability of the KCa channels increased from a control level of 0.08 ± 0.03 to 0.17 ± 0.05 even at the lowest ADP-R concentration (0.1 μM) studied. However, cADP-R reduced the KCa-channel activity, and the threshold concentration of cADP-R that decreased the average channel activity of the KCa channels was 1 μM. These results provide evidence that cADP-R is produced and metabolized in the coronary arterial smooth muscle and that a cADP-R/ADP-R pathway participates in the control of the KCa-channel activity in vascular SMC.

Sign in / Sign up

Export Citation Format

Share Document