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Translational Vision Science &... Sep 2021To develop a method to label proliferating corneal endothelial cells (ECs) in rabbits in vivo and track their migration over time.
PURPOSE
To develop a method to label proliferating corneal endothelial cells (ECs) in rabbits in vivo and track their migration over time.
METHODS
We compared intraperitoneal (IP) and intracameral (IC) administration of 5-ethynyl-2'-deoxyuridine (EdU) in two experiments: (1) six rabbits received IP or IC EdU. Blood and aqueous humor (AH) samples were incubated with HL-60 cells. Flow cytometry detected the EdU incorporation, representing the bioavailability of EdU. (2) In vivo EdU labeling was investigated in pulse-chase study: 48 rabbits received EdU IP or IC. The corneas were flat-mounted after 1, 2, 5, or 40 days and imaged using fluorescence microscopy. EdU+ and Ki67+ ECs were quantified and their distance from the peripheral endothelial edge was measured.
RESULTS
EdU was bioavailable in the AH up to 4 hours after IC injection. No EdU was detected in the blood or the AH after IP injection. High quality EdU labeling of EC was obtained only after IC injection, achieving 2047 ± 702 labeled ECs. Proliferating ECs were located exclusively in the periphery within 1458 ± 146 µm from the endothelial edge. After 40 days, 1490 ± 397 label-retaining ECs (LRCs) were detected, reaching 2219 ± 141 µm from the edge, indicating that LRCs migrated centripetally.
CONCLUSIONS
IC EdU injection enables the labeling and tracking of proliferating ECs. LRCs seem to be involved in endothelial homeostasis, yet it remains to be investigated whether they represent endothelial progenitor cells.
TRANSLATIONAL RELEVANCE
EdU labeling in animal models can aid the search for progenitor cells and the development of cell therapy for corneal endothelial dysfunction.
Topics: Animals; Deoxyuridine; Endothelial Cells; Flow Cytometry; Rabbits; Stem Cells
PubMed: 34478491
DOI: 10.1167/tvst.10.11.7 -
BioTechniques Dec 2022This study sought to develop a short DNA detection method using a deoxyuridine probe and polymerase chain reaction. The probe was hybridized to the target short DNA,...
This study sought to develop a short DNA detection method using a deoxyuridine probe and polymerase chain reaction. The probe was hybridized to the target short DNA, which was then extended by DNA polymerase. The extended DNA was used for real-time PCR after the probe was removed by uracil DNA glycosylase. This method measured from 0.01 to 10 nM of a model short DNA sequence of 17 nucleotides. The method was then used to detect the nucleic acid medicine fomivirsen, as well as 21 phosphorothioate nucleotides, and to quantify 0.1-100 nM of fomivirsen. This method may be useful for detecting short DNA fragments, such as functional nucleotides.
Topics: Real-Time Polymerase Chain Reaction; DNA; Thionucleotides; Deoxyuridine
PubMed: 36350046
DOI: 10.2144/btn-2022-0068 -
The American Journal of Clinical... Apr 2015Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an...
BACKGROUND
Folic acid prevents neural tube closure defects (NTDs), but the causal metabolic pathways have not been established. Serine hydroxymethyltransferase 1 (SHMT1) is an essential scaffold protein in folate-dependent de novo thymidylate synthesis in the nucleus. SHMT1-deficient mice provide a model to investigate folic acid-responsive NTDs wherein disruption of de novo thymidylate synthesis impairs neural tube closure.
OBJECTIVE
We examined the effects of maternal supplementation with the pyrimidine nucleosides uridine, thymidine, or deoxyuridine with and without folate deficiency on NTD incidence in the Shmt1 mouse model.
DESIGN
Shmt1(+/+) and Shmt1(-/-) female mice fed folate-replete or folate-deficient diets and supplemented with uridine, thymidine, or deoxyuridine were bred, and litters (n = 10-23 per group) were examined for the presence of NTDs. Biomarkers of impaired folate status and metabolism were measured, including plasma nucleosides, hepatic uracil content, maternal plasma folate concentrations, and incorporation of nucleoside precursors into DNA.
RESULTS
Shmt1(+/-) and Shmt1(-/-) embryos from dams fed the folate-deficient diet were susceptible to NTDs. No NTDs were observed in litters from dams fed the folate-deficient diet supplemented with deoxyuridine. Surprisingly, uridine supplementation increased NTD incidence, independent of embryo genotype and dietary folic acid. These dietary nucleosides did not affect maternal hepatic uracil accumulation in DNA but did affect plasma folate concentrations.
CONCLUSIONS
Maternal deoxyuridine supplementation prevented NTDs in dams fed the folate-deficient diet, whereas maternal uridine supplementation increased NTD incidence, independent of folate and embryo genotype. These findings provide new insights into the metabolic impairments and mechanisms of folate-responsive NTDs resulting from decreased Shmt1 expression.
Topics: Animals; Deoxyuridine; Disease Models, Animal; Female; Folic Acid; Folic Acid Deficiency; Glycine Hydroxymethyltransferase; HeLa Cells; Humans; Maternal Nutritional Physiological Phenomena; Mice; Neural Tube; Neural Tube Defects; Pregnancy; Thymidine; Uracil; Uridine
PubMed: 25833982
DOI: 10.3945/ajcn.114.097279 -
F&S Science Aug 2021To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts.
OBJECTIVE
To measure the influence of exogenous insulin-like growth factor 1 (IGF1) on follicle growth and maturation in human ovarian cortical xenografts.
DESIGN
Xenotransplantation model.
SETTING
University-based research laboratory.
PATIENTS/ANIMALS
Ovarian tissue was donated with consent and institutional review board approval by brain-dead organ donors or patients undergoing ovarian tissue cryopreservation for fertility preservation. Cortical fragments were transplanted into immunocompromised mice.
INTERVENTIONS
Cryopreserved ovarian cortical fragments from four women (aged 19, 25, 33, and 46 years) were transplanted into the gluteus muscle of immunocompromised mice in a fibrin matrix containing endothelial cells that were transduced with lentiviral particles encoding secreted IGF1. Xenografts were recovered after 3, 8, and 14 weeks. In addition, C57/Bl6 mice underwent intraovarian injection of saline or recombinant IGF1 (60 μg), followed by superovulation, analysis of ethynyl-deoxyuridine incorporation, and ribonucleic acid sequencing of the whole ovaries.
MAIN OUTCOME MEASURES
For xenografts: follicle count and distribution; antral follicle count; and corpora lutea/albicans count. For mice: follicle count and distribution; oocyte yield, ethynyl-deoxyuridine incorporation (granulosa cell proliferation); and ovarian transcriptomic signature.
RESULTS
At 3 weeks, xenografts in the IGF1 condition revealed a decreased percentage of primary follicles and increased percentage of secondary follicles that were concentrated in the preantral subtype; at 8 weeks, an increase in secondary follicles was concentrated in the simple subtype; after 14 weeks, primordial follicles were reduced, and while the number of advanced follicles did not power the experiment to demonstrate significance, antral follicles reduced and corpora lutea increased. Supporting experiments in mice revealed an increase in normal oocytes following intraovarian injection of recombinant IGF1 (60 μg) as well as increased proliferative index among follicles of secondary and preantral stages. Ribonucleic acid sequencing analysis of the whole ovaries following injection of recombinant IGF1 (25 μg) revealed an acute (24 hours) upregulation of transcripts related to steroidogenesis and luteinization.
CONCLUSIONS
Exogenous IGF1 advances the pace of growth among primordial, primary, and secondary stage follicles but results in near absence of antral stage follicles in long-term (14 weeks) xenografts. In mice, acute administration of IGF1 promotes follicle advance and increased oocyte yield. The results suggest that while superphysiological IGF1 alone advances the pace of growth among early/preantral follicles, a sustained and/or later-stage influence undermines antral follicle growth/survival or promotes premature luteinization. These findings provide a temporal framework for interpreting follicle growth/mobilization and may be useful in understanding the clinical application of human growth hormone in the context of assisted reproduction.
Topics: Animals; Deoxyuridine; Endothelial Cells; Female; Heterografts; Humans; Insulin-Like Growth Factor I; Mice; Ovary; RNA; Transplantation, Heterologous
PubMed: 35560275
DOI: 10.1016/j.xfss.2021.07.002 -
The FEBS Journal Jan 2020Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch...
Streptococcus pneumoniae is a Gram-positive bacterium that is a major agent of community-acquired bacterial pneumonia, meningitis and sepsis. Although the mismatch repair function of S. pneumoniae has been assigned to the hexA-hexB gene products, an enzyme capable of the direct elimination of noncanonical nucleotides from the cytoplasm has not been described for this bacterium. Our results show that Spr1057, a protein with previously unknown function, is involved in the inactivation of mutagenic pyrimidine nucleotides and was accordingly designated PynA (pyrimidine nucleotidase A). Biochemical assays confirmed the phosphatase activity of the recombinant enzyme and revealed its metal ion dependence for optimal enzyme activity. We demonstrated that PynA forms a homodimer with higher in vitro activity towards noncanonical 5-fluoro-2'-deoxyuridine monophosphate than towards canonical thymidine monophosphate. Furthermore, we showed via in vivo assays that PynA protects cells against noncanonical pyrimidine derivatives such as 5-fluoro-2'-deoxyuridine and prevents the incorporation of the potentially mutagenic 5-bromo-2'-deoxyuridine (5-BrdU) into DNA. Fluctuation analysis performed under S. pneumoniae exposure to 5-BrdU revealed that the pynA null strain accumulates random mutations with high frequency, resulting in a 30-fold increase in the mutation rate. The data support a model in which PynA, a protein conserved in other Gram-positive bacteria, functions as a house-cleaning enzyme by selectively eliminating noncanonical nucleotides and maintaining the purity of dNTP pools, similar to the YjjG protein described for Escherichia coli.
Topics: 5'-Nucleotidase; Bacterial Proteins; Cations; Deoxyuridine; Mutation Rate; Streptococcus pneumoniae; Substrate Specificity; Thymidine Monophosphate
PubMed: 31437335
DOI: 10.1111/febs.15049 -
PloS One 2015The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part...
BACKGROUND
The kidney is a specialized low-regenerative organ with several different types of cellular lineages. The BrdU label-retaining cell (LRCs) approach has been used as part of a strategy to identify tissue-specific stem cells in the kidney; however, because the complementary base pairing in double-stranded DNA blocks the access of the anti-BrdU antibody to BrdU subunits, the stem cell marker expression in BrdU-labeled cells are often difficult to detect. In this study, we introduced a new cell labeling and detection method in which BrdU was replaced with 5-ethynyl-2-deoxyuridine (EdU) and examined the time-dependent dynamic changes of EdU-labeled cells and potential stem/progenitor markers in the development of kidney.
METHODS
Newborn rats were intraperitoneally injected with EdU, and their kidneys were harvested respectively at different time points at 1 day, 3 days, 1 week, 2 weeks, and 6 weeks post-injection. The kidney tissues were processed for EdU and cellular markers by immunofluorescence staining.
RESULTS
At the early stage, LRCs labeled by EdU were 2176.0 ± 355.6 cells at day one in each renal tissue section, but dropped to 168 ± 48.4 cells by week 6. As time increased, the numbers of LRCs were differentially expressed in the renal cortex and papilla. At the postnatal day one, nearly twice as many cells in the cortex were EdU-labeled as compared to the papilla (28.6 ± 3.6% vs. 15.6 ± 3.4%, P<0.05), while there were more LRCs within the renal papilla since the postnatal week one, and at the postnatal week 6, one third as many cells in the cortex were EdU-labeled as compared to the papilla (2.5 ± 0.1% vs. 7.7 ± 2.7%, P<0.05). The long-term LRCs at 6-week time point were associated exclusively with the glomeruli in the cortex and the renal tubules in the papilla. At 6 weeks, the EdU-labeled LRCs combined with expression of CD34, RECA-1, Nestin, and Synaptopodin were discretely but widely distributed within the glomeruli; Stro-1 around the glomeruli; and α-smooth muscle actin (SMA) in arteries. Conversely, co-expression of CD34, RECA-1, and Nestin with the long term EdU-labeled LRCs was significantly lower in renal tubules (P<0.01), while Stro-1 and Synaptopodin were not detected.
CONCLUSION
Our data found that at 6-week time point, EdU-labeled LRCs existing in the glomeruli expressed undifferentiated podocyte and endothelial markers at high rates, while those in the renal tubules expressed Nestin and vascular markers at low rates. To understand the characterization and localization of these EdU-LRCs, further studies will be needed to test cell lineage tracing, clonogenicity and differentiation potency, and the contributions to the regeneration of the kidney in response to renal injury/repair.
Topics: Animals; Biomarkers; Bromodeoxyuridine; Cell Differentiation; Cell Lineage; Cell Proliferation; Deoxyuridine; Fluorescent Antibody Technique; Image Processing, Computer-Assisted; Kidney; Kinetics; Male; Organogenesis; Rats; Rats, Sprague-Dawley; Staining and Labeling; Stem Cells
PubMed: 26650841
DOI: 10.1371/journal.pone.0144734 -
Proceedings of the National Academy of... Aug 2022Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such...
Nucleotide excision repair is the principal mechanism for removing bulky DNA adducts from the mammalian genome, including those induced by environmental carcinogens such as UV radiation, and anticancer drugs such as cisplatin. Surprisingly, we found that the widely used thymidine analog EdU is a substrate for excision repair when incorporated into the DNA of replicating cells. A number of thymidine analogs were tested, and only EdU was a substrate for excision repair. EdU excision was absent in repair-deficient cells, and in vitro, DNA duplexes bearing EdU were also substrates for excision by mammalian cell-free extracts. We used the excision repair sequencing (XR-seq) method to map EdU repair in the human genome at single-nucleotide resolution and observed that EdU was excised throughout the genome and was subject to transcription-coupled repair as evidenced by higher repair rates in the transcribed strand (TS) relative to the nontranscribed strand (NTS) in transcriptionally active genes. These properties of EdU, combined with its cellular toxicity and ability to cross the blood-brain barrier, make it a potential candidate for treating cancers of the brain, a tissue that typically demonstrates limited replication in adults.
Topics: DNA; DNA Damage; DNA Repair; Deoxyuridine; Genome, Human; Humans; Thymidine; Transcription, Genetic; Ultraviolet Rays
PubMed: 35994676
DOI: 10.1073/pnas.2210176119 -
Advances in Pharmacology and... 1975Computed tomographic (CT) and surgical findings were correlated retrospectively in 51 patients with preoperative diagnoses of prolactin-secreting pituitary... (Review)
Review
Computed tomographic (CT) and surgical findings were correlated retrospectively in 51 patients with preoperative diagnoses of prolactin-secreting pituitary microadenomas. Twenty-four had microadenomas at surgery. Twenty-eight had identifiable discrete lesions. Of these, 18 had microadenomas and 10 did not; these two groups could not be distinguished reliably. Six patients with proven microadenomas had normal CT scans. Focal hypodense lesions, sellar floor erosion, infundibulum displacement, gland height greater than 8 mm, and an abnormal diaphragma sellae configuration are neither sensitive nor specific findings of microadenoma. A significant number of patients with proven microadenomas had few or none of these abnormalities. Thus, recognition of prolactin microadenoma is seldom possible by CT alone, even with high-resolution direct coronal imaging.
Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Antiviral Agents; Carcinogens; Cell Transformation, Neoplastic; DNA Nucleotidyltransferases; Deoxyuridine; Humans; Interferons; Mice; Oncogenic Viruses; Plant Extracts; Polymers; RNA Viruses; Reverse Transcriptase Inhibitors; Viral Vaccines; Virus Diseases
PubMed: 50721
DOI: 10.1016/s1054-3589(08)60220-3 -
Nature Protocols Feb 2021The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental...
The ability to monitor DNA replication fork directionality at the genome-wide scale is paramount for a greater understanding of how genetic and environmental perturbations can impact replication dynamics in human cells. Here we describe a detailed protocol for isolating and sequencing Okazaki fragments from asynchronously growing mammalian cells, termed Okazaki fragment sequencing (Ok-seq), for the purpose of quantitatively determining replication initiation and termination frequencies around specific genomic loci by meta-analyses. Briefly, cells are pulsed with 5-ethynyl-2'-deoxyuridine (EdU) to label newly synthesized DNA, and collected for DNA extraction. After size fractionation on a sucrose gradient, Okazaki fragments are concentrated and purified before click chemistry is used to tag the EdU label with a biotin conjugate that is cleavable under mild conditions. Biotinylated Okazaki fragments are then captured on streptavidin beads and ligated to Illumina adapters before library preparation for Illumina sequencing. The use of Ok-seq to interrogate genome-wide replication fork initiation and termination efficiencies can be applied to all unperturbed, asynchronously growing mammalian cells or under conditions of replication stress, and the assay can be performed in less than 2 weeks.
Topics: Click Chemistry; DNA; DNA Replication; Deoxyuridine; High-Throughput Nucleotide Sequencing; Humans; Streptavidin
PubMed: 33442052
DOI: 10.1038/s41596-020-00454-5 -
Biochemistry Sep 2023The enzyme 2'-deoxynucleoside 5'-phosphate -hydrolase 1 (DNPH1) catalyzes the -ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate...
The enzyme 2'-deoxynucleoside 5'-phosphate -hydrolase 1 (DNPH1) catalyzes the -ribosidic bond cleavage of 5-hydroxymethyl-2'-deoxyuridine 5'-monophosphate to generate 2-deoxyribose 5-phosphate and 5-hydroxymethyluracil. DNPH1 accepts other 2'-deoxynucleoside 5'-monophosphates as slow-reacting substrates. DNPH1 inhibition is a promising strategy to overcome resistance to and potentiate anticancer poly(ADP-ribose) polymerase inhibitors. We solved the crystal structure of unliganded human DNPH1 and took advantage of the slow reactivity of 2'-deoxyuridine 5'-monophosphate (dUMP) as a substrate to obtain a crystal structure of the DNPH1:dUMP Michaelis complex. In both structures, the carboxylate group of the catalytic Glu residue, proposed to act as a nucleophile in covalent catalysis, forms an apparent low-barrier hydrogen bond with the hydroxyl group of a conserved Tyr residue. The crystal structures are supported by functional data, with liquid chromatography-mass spectrometry analysis showing that DNPH1 incubation with dUMP leads to slow yet complete hydrolysis of the substrate. A direct UV-vis absorbance-based assay allowed characterization of DNPH1 kinetics at low dUMP concentrations. A bell-shaped pH-rate profile indicated that acid-base catalysis is operational and that for maximum /, two groups with an average p of 6.4 must be deprotonated, while two groups with an average p of 8.2 must be protonated. A modestly inverse solvent viscosity effect rules out diffusional processes involved in dUMP binding to and possibly uracil release from the enzyme as rate limiting to /. Solvent deuterium isotope effects on / and were inverse and unity, respectively. A reaction mechanism for dUMP hydrolysis is proposed.
Topics: Humans; Hydrolysis; Catalysis; Solvents; Deoxyuridine; Hydrolases; Phosphates; Kinetics; Hydrogen-Ion Concentration
PubMed: 37582341
DOI: 10.1021/acs.biochem.3c00369