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Newly regenerated dopaminergic neurons in 6-OHDA-lesioned adult zebrafish brain proliferate in the Olfactory bulb and telencephalon, but migrate to, differentiate and mature in the diencephalon

Vijayanathan Y, Hamzah NM, Lim SM, Lim FT, Tan MP, Majeed ABA, et al.

Brain Res Bull, 2022 Nov;190:218-233.
PMID: 36228872 DOI: 10.1016/j.brainresbull.2022.10.001

In order to understand the biological processes underlying dopaminergic neurons (DpN) regeneration in a 6-hydroxydopamine(6-OHDA)-induced adult zebrafish-based Parkinson's disease model, this study investigated the specific phases of neuroregeneration in a time-based manner. Bromodeoxyuridine (BrdU) was administered 24 h before the harvest of brain tissues at day three, five, seven, nine, 12 and 14 postlesion. Potential migration of proliferative cells was tracked over 14 days postlesion through double-pulse tracking [BrdU and 5-ethynyl-2'-deoxyuridine (EdU)] of cells and immunohistostaining of astrocytes [glial fibrillary acidic protein (GFAP)]. Gene expression of foxa2 and nurr1 (nr4a2a) at day three, nine, 14, 18, 22 and 30 postlesion was quantified using qPCR. Protein expression of foxa2 at day three, seven, 14 and 22 postlesion was validated using the western blot technique. Double labelling [EdU and tyrosine hydroxylase (TH)] of proliferative cells was performed to ascertain their fate after the neuroregeneration processes. It was found that whilst cell proliferation remained unchanged in the area of substantial DpN loss, the ventral diencephalon (vDn), there was a transient increase of cell proliferation in the olfactory bulb (OB) and telencephalon (Tel) seven days postlesion. BrdU-immunoreactive (ir)/ EdU-ir cells and activated astrocytes were later found to be significantly increased in the vDn and its nearby area (Tel) 14 days postlesion. There was a significant but transient downregulation of foxa2 at day three and nine postlesion, and nr4a2a at day three, nine and 14 postlesion. The expression of both genes remained unchanged in the OB and Tel. There was a transient downregulation of foxa2 protein expression at day three and seven postlesion. The significant increase of EdU-ir/ TH-ir cells in the vDn 30 days postlesion indicates maturation of proliferative cells (formed between day five-seven postlesion) into DpN. The present findings warrant future investigation of critical factors that govern the distinctive phases of DpN regeneration.

Matched MeSH terms: Bromodeoxyuridine/metabolism
Fulltext Evaluation of in vitro wound healing efficacy of breadfruit derived starch hydrolysate

Amin, Z.M., Koh, S.P., Tan, C.P., Yeap, S.K., Hamid, N.S.A., Long, K.

International Food Research Journal, 2017;24(4):1771-1781.
MyJurnal

To study the wound healing efficacy of breadfruit starch hydrolysate, an in vitro wound scratch assay was conducted, in which the migration rate of wounded NIH 3T3 fibroblasts was determined. Wounds treated with lower dextrose equivalent (DE), (DE 10-14) starch hydrolysate were found capable to improve the wound healing of NIH 3T3 fibroblast cell with the percentage of wound closure improvement of 77%, respectively when compared with higher DE range (DE 15-19 and DE 20-24). The findings obtained in the BrdU uptake and MTT viability assays confirmed the wound healing properties of breadfruit starch hydrolysate as the starch hydrolysate-treated wounded NIH 3T3 fibroblasts were able to proliferate well and no cytotoxicity was observed. Together, these findings indicated that the newly developed breadfruit starch hydrolysate performed better than commercial (COM) starch hydrolysate of the same DE ranges. In conclusion, breadfruit starch hydrolysate had better functional properties than did starch hydrolysates derived from other sources and that they could play a beneficial role in wound healing applications.

Matched MeSH terms: Bromodeoxyuridine
Fulltext BrdU pulse labelling in vivo to characterise cell proliferation during regeneration and repair following injury to the airway wall in sheep

Yahaya B, McLachlan G, Collie DD

ScientificWorldJournal, 2013;2013:871932.
PMID: 23533365 DOI: 10.1155/2013/871932

The response of S-phase cells labelled with bromodeoxyuridine (BrdU) in sheep airways undergoing repair in response to endobronchial brush biopsy was investigated in this study. Separate sites within the airway tree of anaesthetised sheep were biopsied at intervals prior to pulse labelling with BrdU, which was administered one hour prior to euthanasia. Both brushed and spatially disparate unbrushed (control) sites were carefully mapped, dissected, and processed to facilitate histological analysis of BrdU labelling. Our study indicated that the number and location of BrdU-labelled cells varied according to the age of the repairing injury. There was little evidence of cell proliferation in either control airway tissues or airway tissues examined six hours after injury. However, by days 1 and 3, BrdU-labelled cells were increased in number in the airway wall, both at the damaged site and in the regions flanking either side of the injury. Thereafter, cell proliferative activity largely declined by day 7 after injury, when consistent evidence of remodelling in the airway wall could be appreciated. This study successfully demonstrated the effectiveness of in vivo pulse labelling in tracking cell proliferation during repair which has a potential value in exploring the therapeutic utility of stem cell approaches in relevant lung disease models.

Matched MeSH terms: Bromodeoxyuridine/chemistry
Association between apoptotic neural tissue and cell proliferation in the adult teleost brain

Lim FT, Ogawa S, Parhar IS

Brain Res, 2016 11 01;1650:60-72.
PMID: 27568467 DOI: 10.1016/j.brainres.2016.08.033

Injury to neuronal tissues in the central nervous system (CNS) of mammals results in neural degeneration and sometime leads to loss of function, whereas fish retain a remarkable potential for neuro-regeneration throughout life. Thus, understanding the mechanism of neuro-regeneration in fish CNS would be useful to improve the poor neuro-regenerative capability in mammals. In the present study, we characterized a neuro-regenerative process in the brain of a cichlid, tilapia, Oreochromis niloticus. Morphological observations showed that the damaged brain region (habenula) successfully regrew and reinnervated axonal projections by 60 days post-damage. A fluorescent carbocyanine tracer, DiI tracing revealed a recovery of the major neuronal projection from the regenerated habenula to the interpenduncular nucleus by 60 days post-damage. TUNEL assay showed a significant increase of apoptotic cells (~234%, P<0.01) at one day post-damage, while the number of bromodeoxyuridine (BrdU)-positive proliferative cells were significantly increased (~92%, P<0.05) at 7 days post-damage compared with sham-control fish. To demonstrate a potential role of apoptotic activity in the neuro-regeneration, effects of degenerative neural tissue on cell proliferation were examined in vivo. Implantation of detached neural but not non-neural tissues into the cranial cavity significantly (P<0.01) increased the number of BrdU-positive cells nearby the implantation regions at 3 days after the implantation. Furthermore, local injection of the protein extract and cerebrospinal fluid collected from injured fish brain significantly induced cell proliferation in the brain. These results suggest that factor(s) derived from apoptotic neural cells may play a critical role in the neuro-regeneration in teleost brain.

Matched MeSH terms: Bromodeoxyuridine/metabolism
Fulltext Cytotoxicity of leukemic cells by nypa fruticans through regulation of adiponectin expression

Mohammad Fauzan Zainudin, Ummu Afifah Fadzir, Athirah Rosdi, Muhammad Farid Johan, Ridzwan Hashim, Ridhwan Abdul Wahab, et al.

International Journal of Allied Health Sciences, 2017;1(2):28-43.
MyJurnal

Acute lymphoblastic leukemia (ALL) is the most common leukemia subtypes among paediatrics in Malaysia. Although treatment options are available but some patients remain incurable, some undergo relapse and many experiences adverse effects by the conventional therapies. Thus, we aim to investigate possible treatment alternative by studying the antileukemogenesis properties of concentrated Nypa fruticans sap called nisaan by focusing on adiponectin expression.
Our study model was CCRF-CEM, an acute lymphoblastic leukemia cell lines. The cells were treated with nisaan at a range of concentration and treated for 24, 48 and 72 hours followed by determination of the leukemic cells viability using tryphan blue method. Effective nisaan concentrations that significantly reduced the cells viability were again treated to the cells followed by determination of the cell proliferation using BrdU colorimetric kit and adiponectin level using adiponectin ELISA kit.
The results showed that, increase concentration of nisaan treatment reduced the cells viability and cells proliferation and enhance the adiponectin level in the leukemic cells.
This preliminary data suggest that Nypa fruticans might has the antileukemogenesis effect on acute lymphoblastic cells by regulating the adiponectin expression.

Matched MeSH terms: Bromodeoxyuridine
Endothelial replicative senescence delayed by the inhibition of MTORC1 signaling involves MicroRNA-107

Khor ES, Wong PF

Int J Biochem Cell Biol, 2018 Aug;101:64-73.
PMID: 29857052 DOI: 10.1016/j.biocel.2018.05.016

Accumulation of senescent endothelial cells can contribute to endothelium dysfunction. Suppression of MTOR signaling has been shown to delay senescence but the mechanism that underpins this effect, particularly one that involves miRNAs, remains to be further defined. This study sought to identify miRNAs involved in MTORC1-mediated inhibition of replicative senescence in endothelial cells. Pre-senescent HUVECs were prolonged treated with low dose rapamycin (1 nM), an MTOR inhibitor. Rapamycin treatment down-regulated the phosphorylated MTOR, RPS6 and 4EBP1 expressions, which confirmed MTORC1 suppression. Prolonged low dose rapamycin treatment has significantly reduced the percentage of senescence-associated beta galactosidase (SA-β gal) positively stained senescent cells and P16INK4A expression in these cells. On the contrary, the percentage of BrdU-labelled proliferating cells has significantly increased. RPTOR, a positive regulator of MTORC1 was knockdown using RPTOR siRNA to inhibit MTORC1 activation. RPTOR knockdown was evidenced by significant suppressions of RPTOR mRNA and protein expression levels. In these cells, the expression of miR-107 was down-regulated whereas miR-145-5p and miR-217 were up-regulated. Target gene prediction revealed PTEN as the target of miR-107 and this was confirmed by biotin pull-down assay. Over-expression of miR-107 has decreased PTEN expression, increased MTORC1 activity, induced cell cycle arrest at G0/G1 phase and up-regulated P16INK4A expression but mitigated tube formation. Collectively, our findings revealed that delayed endothelial replicative senescence caused by the inhibition of MTORC1 activation could be modulated by miR-107 via its influence on PTEN.

Matched MeSH terms: Bromodeoxyuridine
Fulltext Betulinic Acid inhibits growth of cultured vascular smooth muscle cells in vitro by inducing g(1) arrest and apoptosis

Vadivelu RK, Yeap SK, Ali AM, Hamid M, Alitheen NB

Evid Based Complement Alternat Med, 2012;2012:251362.
PMID: 23056140 DOI: 10.1155/2012/251362

Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC) is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC(50) of 3.8 μg/mL significantly (P < 0.05). Nevertheless, betulinic acid exhibited G(1) cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G(1) cell cycle arrest and dose-dependent DNA damage on VSMC.

Matched MeSH terms: Bromodeoxyuridine
Genomic variations among wildtype and mutant strains of pseudorabies virus

Zeenathul NA, Mohd-Azmi ML, Ali AS, Aini I, Sheik-Omar AR, Abdul-Rahim AM, et al.

Rev. Argent. Microbiol., 2002 Jan-Mar;34(1):7-14.
PMID: 11942085

Both wild-type virulent and mutant strains of pseudorabies virus (PrV) were used in this study. Mutants used were derived from the plaque purified strain PrVmAIP. A total of six drug resistant mutants, three bromodeoxyuridine (BUdR) resistant and three iododeoxyuridine (IUdR) resistant, respectively, were isolated and passaged in chicken embryo fibroblast (CEF) cells. The DNA of these PrVs were compared with the wild-type isolates by means of the restriction fragment pattern (RFP) findings produced with Bam HI, Kpn I, Hind III and Bgl II restriction enzymes (RE). Compared to the wild-type PrVs (PrV-VBA1-parental strain of PrVmAIP; PrV-VBA2; PrV-VBA3), the RFP of PrVmAIP showed the presence of mutations within the RE sites studied. Both PrV-VBA1 and PrV-VBA2 appeared to be closely related but their RFPs differed from PrV-VBA3. Significant differences either in the number, size or migrations of the DNA fragments could also be detected in the BUdR resistant strains. Even though different features of cytopathic effect (GPE) were observed in the IUdR resistant PrVs, the RFP findings remained identical. The PrVs studied showed considerable differences from the reference PrV (PrV-CD).

Matched MeSH terms: Bromodeoxyuridine/pharmacology
Fulltext Selective cytotoxicity effects of cytobiotics produced by lactic acid bacteria isolated from Malaysian foods

Hooi Ling Foo

Malaysian Journal of Medicine and Health Sciences, 2019;15(108):7-7.
MyJurnal

Probiotics are live microorganisms and when consumed in adequate amounts will confer health benefit on the host. Probiotic effects of Lactic Acid Bacteria (LAB) have been reported extensively, which rely generally on the viability of LAB cells. However, we have reported extensively the prominent probiotic effects of cell less postbiotics metabolites produced by various strains of Lactobacillus plantarum isolated from Malaysian foods on rats, poultry and pigs. L. plantarum is a major species of LAB. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on the cytotoxic and antiproliferative activities of cytobiotic metabolites produced by LAB. Recently, we have documented the selective antiproliferative and cytotoxicity of cytobiotic produced by six strains of L. plantarum on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. Haemolytic assay was used to determine the toxicity of cytobiotic using human and various animal red blood cells. The cytotoxicity mode was subsequently determined for selected UL4 cytobiotic on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic ob-servation using AO/PI dye reagents and flow cytometric analyses. The selective cytotoxicity effect on various cancel cells that occurred in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells will be discussed in the presentation. Moreover, the antiproliferative effects and induction of late apoptosis effects against selected malignant cancer cells will be discussed further in the presentation. This report reveals the vast potential of cytobiotics produced by L. plantarum strains as functional supplement and as an adjunctive treatment for cancer.

Matched MeSH terms: Bromodeoxyuridine
Goniothalamin induces apoptosis in vascular smooth muscle cells

Chan KM, Rajab NF, Ishak MH, Ali AM, Yusoff K, Din LB, et al.

Chem Biol Interact, 2006 Feb 1;159(2):129-40.
PMID: 16297902

Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.

Matched MeSH terms: Bromodeoxyuridine
Fulltext In vitro and in vivo antitumour effects of coconut water vinegar on 4T1 breast cancer cells

Mohamad NE, Yeap SK, Abu N, Lim KL, Zamberi NR, Nordin N, et al.

Food Nutr Res, 2019;63.
PMID: 30814922 DOI: 10.29219/fnr.v63.1616

Background: Coconut water and vinegars have been reported to possess potential anti-tumour and immunostimulatory effects. However, the anti-tumour, anti-inflammatory and immunostimulatory effects of coconut water vinegar have yet to be tested.
Objective: This study investigated the in vitro and in vivo anti-tumour effects of coconut water vinegar on 4T1 breast cancer cells.
Methods: The 4T1 cells were treated with freeze-dried coconut water vinegar and subjected to MTT cell viability, BrdU, annexin V/PI apoptosis, cell cycle and wound healing assays for the in vitro analysis. For the in vivo chemopreventive evaluation, mice challenged with 4T1 cells were treated with 0.08or 2.00 mL/kg body weight of fresh coconut water vinegar for 28 days. Tumour weight, apoptosis of tumour cells, metastasis and immunity of untreated mice and coconut water vinegar-treated 4T1 challenged mice were compared.
Results: Freeze-dried coconut water vinegar reduced the cell viability, induced apoptosis and delayed the wound healing effect of 4T1 cells in vitro. In vivo, coconut water vinegar delayed 4T1 breast cancer progression in mice by inducing apoptosis and delaying the metastasis. Furthermore, coconut water vinegar also promoted immune cell cytotoxicity and production of anticancer cytokines. The results indicate that coconut water vinegar delays breast cancer progression by inducing apoptosis in breast cancer cells, suppressing metastasis and activating anti-tumour immunity.
Conclusion: Coconut water vinegar is a potential health food ingredient with a chemopreventive effect.

Matched MeSH terms: Bromodeoxyuridine
Immunomodulatory effects of Potentilla indica and Dendrophthoe pentandra on mice splenocytes and thymocytes

Ang HY, Subramani T, Yeap SK, Omar AR, Ho WY, Abdullah MP, et al.

Exp Ther Med, 2014 Jun;7(6):1733-1737.
PMID: 24926376

Immunomodulators are agents that are able to stimulate or inhibit the immune response. The leaf extracts from Potentilla indica and Dendrophthoe pentandra were analyzed in vitro for immunomodulatory activity and an MTT colorimetric assay was conducted to determine the proliferation of mice splenocytes and thymocytes. A bromodeoxyuridine assay was performed to analyze DNA synthesis and the Trypan blue exclusion method was conducted to evaluate the changes in total cell population. The results indicated that treatment with P. indica and D. pentandra produced a time- and dose-dependent increase in cell viability and proliferation. Following 72 h of treatment with P. indica and D. pentandra, thymocyte proliferation was augmented by 18 and 41%, respectively and splenocyte proliferation increased by 35 and 42%, respectively, when compared with untreated cells. The present study demonstrated that these extracts may act as potential immunostimulants and, thus, represent an alternative source of immunomodulatory compounds for the treatment of human immune-mediated diseases.

Matched MeSH terms: Bromodeoxyuridine
Fulltext The Tocotrienol-Rich Fraction Is Superior to Tocopherol in Promoting Myogenic Differentiation in the Prevention of Replicative Senescence of Myoblasts

Khor SC, Razak AM, Wan Ngah WZ, Mohd Yusof YA, Abdul Karim N, Makpol S

PLoS One, 2016;11(2):e0149265.
PMID: 26885980 DOI: 10.1371/journal.pone.0149265

Aging results in a loss of muscle mass and strength. Myoblasts play an important role in maintaining muscle mass through regenerative processes, which are impaired during aging. Vitamin E potentially ameliorates age-related phenotypes. Hence, this study aimed to determine the effects of the tocotrienol-rich fraction (TRF) and α-tocopherol (ATF) in protecting myoblasts from replicative senescence and promoting myogenic differentiation. Primary human myoblasts were cultured into young and senescent stages and were then treated with TRF or ATF for 24 h, followed by an analysis of cell proliferation, senescence biomarkers, cellular morphology and differentiation. Our data showed that replicative senescence impaired the normal regenerative processes of myoblasts, resulting in changes in cellular morphology, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation and myogenic regulatory factors (MRFs) expression. Treatment with both TRF and ATF was beneficial to senescent myoblasts in reclaiming the morphology of young cells, improved cell viability and decreased SA-β-gal expression. However, only TRF treatment increased BrdU incorporation in senescent myoblasts, as well as promoted myogenic differentiation through the modulation of MRFs at the mRNA and protein levels. MYOD1 and MYOG gene expression and myogenin protein expression were modulated in the early phases of myogenic differentiation. In conclusion, the tocotrienol-rich fraction is superior to α-tocopherol in ameliorating replicative senescence-related aberration and promoting differentiation via modulation of MRFs expression, indicating vitamin E potential in modulating replicative senescence of myoblasts.

Matched MeSH terms: Bromodeoxyuridine/metabolism
Spred-2 expression is associated with neural repair of injured adult zebrafish brain

Lim FT, Ogawa S, Parhar IS

J. Chem. Neuroanat., 2016 11;77:176-186.
PMID: 27427471 DOI: 10.1016/j.jchemneu.2016.07.005

Sprouty-related protein-2 (Spred-2) is a negative regulator of extracellular signal-regulated kinases (ERK) pathway, which is important for cell proliferation, neuronal differentiation, plasticity and survival. Nevertheless, its general molecular characteristics such as gene expression patterns and potential role in neural repair in the brain remain unknown. Thus, this study aimed to characterise the expression of spred-2 in the zebrafish brain. Digoxigenin-in situ hybridization showed spred-2 mRNA-expressing cells were mainly seen in the proliferative zones such as the olfactory bulb, telencephalon, optic tectum, cerebellum, and the dorsal and ventral hypothalamus, and most of which were neuronal cells. To evaluate the potential role of spred-2 in neuro-regeneration, spred-2 gene expression was examined in the dorsal telencephalon followed by mechanical-lesion. Real-time PCR showed a significant reduction of spred-2 mRNA levels in the telencephalon on 1-day till 2-days post-lesion and gradually increased to normal levels as compared with intact. Furthermore, to confirm involvement of Spred-2 signalling in the cell proliferation after brain injury, double-labelling of spred-2 in-situ hybridization with immunofluorescence of BrdU and phosphorylated-ERK1/2 (p-ERK1/2), a downstream of Spred-2 was performed. Increase of BrdU and p-ERK1/2 immunoreactive cells suggest that a decrease in spred-2 after injury might associated with activation of the ERK pathway to stimulate cell proliferation in the adult zebrafish brain. The present study demonstrates the possible role of Spred-2 signalling in cell proliferative phase during the neural repair in the injured zebrafish brain.

Matched MeSH terms: Bromodeoxyuridine/pharmacology
Zinc intake during pregnancy increases the proliferation at ventricular zone of the newborn brain

Azman MS, Wan Saudi WS, Ilhami M, Mutalib MS, Rahman MT

Nutr Neurosci, 2009 Feb;12(1):9-12.
PMID: 19178786 DOI: 10.1179/147683009X388904

Neurogenesis involves cell proliferation, cell cycle arrest, differentiation, migration and the natural developmental death of the neural precursors. These processes are highly co-ordinated and governed by cell-cycle genes and neural transcription factors. Zn plays a crucial role as a functional and structural component of enzymes and transcription factors and components of the intracellular signaling pathway associated with the regulation of cell proliferation. The influence of additional Zn intake during pregnancy on the neuronal proliferation at ventricular zone of the developing fetus has been studied. Pups delivered by the group of mice provided with drinking water with 4.0 mM Zn supplement throughout pregnancy contained an increased number of proliferating neurons in the ventricular zone at P0 compared to those delivered by the mice provided with drinking water without any Zn supplement. This finding provides direct evidence to support the notion that maternal Zn levels influence the development of the nervous system of the offspring.

Matched MeSH terms: Bromodeoxyuridine

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