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1.
Chemistry ; 27(64): 15918-15921, 2021 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-34559417

RESUMO

High fidelity base pairing is important for the transmission of genetic information. Weak base pairs can lower fidelity, complicating sequencing, amplification and replication of DNA. Thymidine 5'-monophosphate (TMP) is the most weakly pairing nucleotide among the canonical deoxynucleotides, causing high errors rates in enzyme-free primer extension. Here we report the synthesis of an ethynylpyridone C-nucleoside analog of 3'-amino-2',3'-dideoxythymidine monophosphate and its incorporation in a growing strand by enzyme-free primer extension. The ethynylpyridone C-nucleotide accelerates extension more than five-fold, reduces misincorporation and readily displaces TMP in competition experiments. The results bode well for the use of the C-nucleoside as replacements for thymidine in practical applications.


Assuntos
DNA , Nucleotídeos , Pareamento de Bases , Primers do DNA , Replicação do DNA , Cinética , Timidina
2.
Molecules ; 26(16)2021 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-34443682

RESUMO

Atherosclerosis is the main cause of cardiovascular diseases which in turn, lead to the highest number of mortalities globally. This pathophysiological condition is developed due to a constant elevated level of plasma cholesterols. Statin is currently the widely used treatment in reducing the level of cholesterols, however, it may cause adverse side effects. Therefore, there is an urgent need to search for new alternative treatment. PCSK9 is an enzyme responsible in directing LDL-receptor (LDL-R)/LDL-cholesterols (LDL-C) complex to lysosomal degradation, preventing the receptor from recycling back to the surface of liver cells. Therefore, PCSK9 offers a potential target to search for small molecule inhibitors which inhibit the function of this enzyme. In this study, a marine invertebrate Acanthaster planci, was used to investigate its potential in inhibiting PCSK9 and lowering the levels of cholesterols. Cytotoxicity activity of A. planci on human liver HepG2 cells was carried out using the MTS assay. It was found that methanolic extract and fractions did not exhibit cytotoxicity effect on HepG2 cell line with IC50 values of more than 30 µg/mL. A compound deoxythymidine also did not exert any cytotoxicity activity with IC50 value of more than 4 µg/mL. Transient transfection and luciferase assay were conducted to determine the effects of A. planci on the transcriptional activity of PCSK9 promoter. Methanolic extract and Fraction 2 (EF2) produced the lowest reduction in PCSK9 promoter activity to 70 and 20% of control at 12.5 and 6.25 µg/mL, respectively. In addition, deoxythymidine also decreased PCSK9 promoter activity to the lowest level of 60% control at 3.13 µM. An in vivo study using Sprague Dawley rats demonstrated that 50 and 100 mg/kg of A. planci methanolic extract reduced the total cholesterols and LDL-C levels to almost similar levels of untreated controls. The level of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) showed that the administration of the extract did not produce any toxicity effect and cause any damage to rat liver. The results strongly indicate that A. planci produced a significant inhibitory activity on PCSK9 gene expression in HepG2 cells which may be responsible for inducing the uptake of cholesterols by liver, thus, reducing the circulating levels of total cholesterols and LDL-C. Interestingly, A. planci also did show any adverse hepato-cytotoxicity and toxic effects on liver. Thus, this study strongly suggests that A. planci has a vast potential to be further developed as a new class of therapeutic agent in lowering the blood cholesterols and reducing the progression of atherosclerosis.


Assuntos
Colesterol/sangue , Pró-Proteína Convertase 9/antagonistas & inibidores , Estrelas-do-Mar/química , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Morte Celular , Proliferação de Células , LDL-Colesterol/sangue , Células Hep G2 , Humanos , Luciferases/metabolismo , Masculino , Metanol , Regiões Promotoras Genéticas/genética , Pró-Proteína Convertase 9/genética , Pró-Proteína Convertase 9/metabolismo , Ratos Sprague-Dawley , Timidina/farmacologia , Triglicerídeos/sangue
3.
Gut Liver ; 15(6): 887-894, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34446613

RESUMO

Background/Aims: To investigate postpartum hepatic flares and associated factors in highly viremic pregnant patients in the immune tolerance phase who adopted telbivudine (LdT) treatment in the last trimester to reduce vertical transmission of hepatitis B virus. Methods: Hepatitis B e antigen (HBeAg)-positive, highly viremic pregnant women were recruited for this prospective study. Treatment with LdT was started from 28 weeks of gestation. Virological and biochemical markers were examined before LdT treatment, antepartum and postpartum. Serial blood samples at the same time were collected to detect cytokines and cortisol (COR). Results: Fifty-six of 153 patients (36.6%) had postpartum hepatic flares, defined as a 2-fold increase in alanine aminotransferase 6 weeks after delivery. Age and the antepartum alanine aminotransferase and postpartum HBeAg levels were independent influencing factors of postpartum hepatic flares. Cytokines showed no regularity during or after pregnancy. Compared with the patients with no postpartum flares, the patients with flares had lower baseline interferon γ and COR levels (p=0.022 and p=0.028) and higher postpartum interferon γ levels (p=0.026). Conclusions: A high proportion of highly viremic and immune-tolerant pregnant patients treated with LdT in the last trimester had postpartum hepatic flares, which implied that these patients entered the immune clearance phase after delivery. Thus, this may create an appropriate opportunity for re-antiviral therapy.


Assuntos
Hepatite B Crônica , Hepatite B , Complicações Infecciosas na Gravidez , Alanina Transaminase , Antivirais/uso terapêutico , DNA Viral , Feminino , Hepatite B/tratamento farmacológico , Antígenos E da Hepatite B , Vírus da Hepatite B/genética , Hepatite B Crônica/tratamento farmacológico , Humanos , Fatores Imunológicos/uso terapêutico , Período Pós-Parto , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Gestantes , Estudos Prospectivos , Telbivudina/uso terapêutico , Timidina
4.
Nucleic Acids Res ; 49(16): 9594-9605, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34390349

RESUMO

Protein evolution has significantly enhanced the development of life science. However, it is difficult to achieve in vitro evolution of some special proteins because of difficulties with heterologous expression, purification, and function detection. To achieve protein evolution via in situ mutation in vivo, we developed a base editor by fusing nCas with a cytidine deaminase in Bacillus subtilis through genome integration. The base editor introduced a cytidine-to-thymidine mutation of approximately 100% across a 5 nt editable window, which was much higher than those of other base editors. The editable window was expanded to 8 nt by extending the length of sgRNA, and conversion efficiency could be regulated by changing culture conditions, which was suitable for constructing a mutant protein library efficiently in vivo. As proof-of-concept, the Sec-translocase complex and bacitracin-resistance-related protein BceB were successfully evolved in vivo using the base editor. A Sec mutant with 3.6-fold translocation efficiency and the BceB mutants with different sensitivity to bacitracin were obtained. As the construction of the base editor does not rely on any additional or host-dependent factors, such base editors (BEs) may be readily constructed and applicable to a wide range of bacteria for protein evolution via in situ mutation.


Assuntos
Bacillus subtilis/genética , Citidina Desaminase/genética , Evolução Molecular , Proteínas/genética , Sistemas CRISPR-Cas/genética , Citidina/genética , Edição de Genes , Genoma Bacteriano/genética , Instabilidade Genômica/genética , Mutação/genética , Timidina/genética
5.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34208896

RESUMO

In this paper, we report our investigations on five T30175 analogues, prepared by replacing sequence thymidines with abasic sites (S) one at a time, in comparison to their natural counterpart in order to evaluate their antiproliferative potential and the involvement of the residues not belonging to the central core of stacked guanosines in biological activity. The collected NMR (Nuclear Magnetic Resonance), CD (Circular Dichroism), and PAGE (Polyacrylamide Gel Electrophoresis) data strongly suggest that all of them adopt G-quadruplex (G4) structures strictly similar to that of the parent aptamer with the ability to fold into a dimeric structure composed of two identical G-quadruplexes, each characterized by parallel strands, three all-anti-G-tetrads and four one-thymidine loops (one bulge and three propeller loops). Furthermore, their antiproliferative (MTT assay) and anti-motility (wound healing assay) properties against lung and colorectal cancer cells were tested. Although all of the oligodeoxynucleotides (ODNs) investigated here exhibited anti-proliferative activity, the unmodified T30175 aptamer showed the greatest effect on cell growth, suggesting that both its characteristic folding in dimeric form and its presence in the sequence of all thymidines are crucial elements for antiproliferative activity. This straightforward approach is suitable for understanding the critical requirements of the G-quadruplex structures that affect antiproliferative potential and suggests its application as a starting point to facilitate the reasonable development of G-quadruplexes with improved anticancer properties.


Assuntos
Antineoplásicos/química , Aptâmeros de Nucleotídeos/química , Neoplasias Colorretais/genética , Neoplasias Pulmonares/genética , Timidina/genética , Substituição de Aminoácidos , Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/genética , Aptâmeros de Nucleotídeos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dicroísmo Circular , Neoplasias Colorretais/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Quadruplex G , Células HCT116 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Espectroscopia de Ressonância Magnética
6.
J Bacteriol ; 203(17): e0015021, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34152201

RESUMO

Bacterial rod-shaped cells experiencing irreparable chromosome damage should filament without other morphological changes. Thymineless death (TLD) strikes thymidine auxotrophs denied external thymine/thymidine (T) supplementation. Such T-starved cells cannot produce the DNA precursor dTTP and therefore stop DNA replication. Stalled replication forks in T-starved cells were always assumed to experience mysterious chromosome lesions, but TLD was recently found to happen even without origin-dependent DNA replication, with the chromosome still remaining the main TLD target. T starvation also induces morphological changes, as if thymidine prevents cell envelope or cytoplasm problems that otherwise translate into chromosome damage. Here, we used transmission electron microscopy (TEM) to examine cytoplasm and envelope changes in T-starved Escherichia coli cells, using treatment with a DNA gyrase inhibitor as a control for "pure" chromosome death. Besides the expected cell filamentation in response to both treatments, we see the following morphological changes specific for T starvation and which might lead to chromosome damage: (i) significant cell widening, (ii) nucleoid diffusion, (iii) cell pole damage, and (iv) formation of numerous cytoplasmic bubbles. We conclude that T starvation does impact both the cytoplasm and the cell envelope in ways that could potentially affect the chromosome. IMPORTANCE Thymineless death is a dramatic and medically important phenomenon, the mechanisms of which remain a mystery. Unlike most other auxotrophs in the absence of the required supplement, thymidine-requiring E. coli mutants not only go static in the absence of thymidine, but rapidly die of chromosomal damage of unclear nature. Since this chromosomal damage is independent of replication, we examined fine morphological changes in cells undergoing thymineless death in order to identify what could potentially affect the chromosome. Here, we report several cytoplasm and cell envelope changes that develop in thymidine-starved cells but not in gyrase inhibitor-treated cells (negative control) that could be linked to subsequent irreparable chromosome damage. This is the first electron microscopy study of cells undergoing "genetic death" due to irreparable chromosome lesions.


Assuntos
Membrana Celular/ultraestrutura , Citoplasma/ultraestrutura , Escherichia coli/metabolismo , Timina/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Replicação do DNA , Escherichia coli/genética , Escherichia coli/ultraestrutura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Viabilidade Microbiana , Microscopia Eletrônica , Timidina/metabolismo
7.
Biomolecules ; 11(5)2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068700

RESUMO

Polyamine spermidine is essential for the proliferation of eukaryotic cells. Administration of polyamine biosynthesis inhibitor α-difluoromethylornithine (DFMO) induces cytostasis that occurs in two phases; the early phase which can be reversed by spermidine, spermine, and some of their analogs, and the late phase which is characterized by practically complete depletion of cellular spermidine pool. The growth of cells at the late phase can be reversed by spermidine and by very few of its analogs, including (S)-1-methylspermidine. It was reported previously (Witherspoon et al. Cancer Discovery 3(9); 1072-81, 2013) that DFMO treatment leads to depletion of cellular thymidine pools, and that exogenous thymidine supplementation partially prevents DFMO-induced cytostasis without affecting intracellular polyamine pools in HT-29, SW480, and LoVo colorectal cancer cells. Here we show that thymidine did not prevent DFMO-induced cytostasis in DU145, LNCaP, MCF7, CaCo2, BT4C, SV40MES13, HepG2, HEK293, NIH3T3, ARPE19 or HT-29 cell lines, whereas administration of functionally active mimetic of spermidine, (S)-1-methylspermidine, did. Thus, the effect of thymidine seems to be specific only for certain cell lines. We conclude that decreased polyamine levels and possibly also distorted pools of folate-dependent metabolites mediate the anti-proliferative actions of DFMO. However, polyamines are necessary and sufficient to overcome DFMO-induced cytostasis, while thymidine is generally not.


Assuntos
Citostáticos/farmacologia , Eflornitina/efeitos adversos , Neoplasias/tratamento farmacológico , Poliaminas/farmacologia , Timidina/farmacologia , Animais , Células Cultivadas , Humanos , Camundongos , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores da Ornitina Descarboxilase/efeitos adversos , Inibidores da Ornitina Descarboxilase/farmacologia
8.
Methods Mol Biol ; 2329: 111-121, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085219

RESUMO

Cell synchronization is crucial when studying events that take place at specific points of the cell cycle. Several chemical agents can be used to achieve the cell culture synchronization but not all type of cells respond equally to a given concentration of these drugs. Here we describe a simple optimization method to select concentrations and timings for nocodazole or thymidine treatments using fluorescence staining. In addition, we provide detailed protocols to arrest an asynchronous culture of either suspension or adherent cells in G1/S or in G2/M.


Assuntos
Técnicas de Cultura de Células/métodos , Nocodazol/farmacologia , Timidina/farmacologia , Adesão Celular , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Corantes Fluorescentes/química , Células HeLa , Humanos , Fatores de Tempo
9.
PLoS One ; 16(4): e0248971, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33793606

RESUMO

Adult growth hormone deficiency (GHD), a condition characterized by increased oxidative stress, is related to augmented cardiovascular, metabolic and oncological risk. A case-control observational study has been performed to evaluate DNA oxidative damage analysing the production of thymidine-glycol in lymphocytes and its correlation with plasma antioxidant levels, evaluated as Total Antioxidant Capacity (TAC). GHD was diagnosed using GHRH 50µg iv+arginine 0,5 g/Kg test, with peak GH response <9 µg/L when BMI was <30 kg/m2 or <4 µg/L when BMI was >30 kg/m2. Three groups were identified: total GHD (n = 16), partial GHD (n = 11), and controls (n = 12). Thymidine-glycol, TAC and IGF-1 have been determined respectively in lymphocytes, plasma and serum samples. When considering thymidine-glycol, we found a significant difference between total vs partial GHD and controls. Unexpectedly thymidine-glycol was lower in total GHD, also accompanied with a significant increase in plasmatic TAC. Our results showed that in adult GHD condition, the production of antioxidant species, in response to increased oxidative stress, could exert a protective effect on thymidine-glycol formation, and consequently on DNA intracellular damages. This pilot study could be inserted in the complex scenario of oxidative damage of GHD, a subtle, yet poorly defined condition, worthy of further insights.


Assuntos
Dano ao DNA , Hormônio do Crescimento Humano/deficiência , Linfócitos/metabolismo , Estresse Oxidativo , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Linfócitos/citologia , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Timidina/análogos & derivados , Timidina/metabolismo
10.
Science ; 372(6538): 156-165, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33833118

RESUMO

Mutations in the BRCA1 or BRCA2 tumor suppressor genes predispose individuals to breast and ovarian cancer. In the clinic, these cancers are treated with inhibitors that target poly(ADP-ribose) polymerase (PARP). We show that inhibition of DNPH1, a protein that eliminates cytotoxic nucleotide 5-hydroxymethyl-deoxyuridine (hmdU) monophosphate, potentiates the sensitivity of BRCA-deficient cells to PARP inhibitors (PARPi). Synthetic lethality was mediated by the action of SMUG1 glycosylase on genomic hmdU, leading to PARP trapping, replication fork collapse, DNA break formation, and apoptosis. BRCA1-deficient cells that acquired resistance to PARPi were resensitized by treatment with hmdU and DNPH1 inhibition. Because genomic hmdU is a key determinant of PARPi sensitivity, targeting DNPH1 provides a promising strategy for the hypersensitization of BRCA-deficient cancers to PARPi therapy.


Assuntos
Antineoplásicos/farmacologia , N-Glicosil Hidrolases/antagonistas & inibidores , N-Glicosil Hidrolases/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Apoptose , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Neoplasias/metabolismo , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Desoxicitidina Monofosfato/farmacologia , Nucleotídeos de Desoxiuracil/metabolismo , Resistencia a Medicamentos Antineoplásicos , Genes BRCA1 , Humanos , Hidrólise , N-Glicosil Hidrolases/genética , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas/genética , Mutações Sintéticas Letais , Timidina/análogos & derivados , Timidina/antagonistas & inibidores , Timidina/metabolismo , Timidina/farmacologia , Uracila-DNA Glicosidase/metabolismo
11.
J Mol Biol ; 433(10): 166949, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33744317

RESUMO

Checkpoint Kinase 1 (Chk1) prevents DNA damage by adjusting the replication choreography in the face of replication stress. Chk1 depletion provokes slow and asymmetrical fork movement, yet the signals governing such changes remain unclear. We sought to investigate whether poly(ADP-ribose) polymerases (PARPs), key players of the DNA damage response, intervene in the DNA replication of Chk1-depleted cells. We demonstrate that PARP inhibition selectively alleviates the reduced fork elongation rates, without relieving fork asymmetry in Chk1-depleted cells. While the contribution of PARPs to fork elongation is not unprecedented, we found that their role in Chk1-depleted cells extends beyond fork movement. PARP-dependent fork deceleration induced mild dormant origin firing upon Chk1 depletion, augmenting the global rates of DNA synthesis. Thus, we have identified PARPs as novel regulators of replication fork dynamics in Chk1-depleted cells.


Assuntos
Quinase 1 do Ponto de Checagem/genética , Replicação do DNA , Poli(ADP-Ribose) Polimerases/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/antagonistas & inibidores , Quinase 1 do Ponto de Checagem/metabolismo , Regulação da Expressão Gênica , Humanos , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Roscovitina/farmacologia , Timidina/análogos & derivados , Timidina/farmacologia
12.
Methods Mol Biol ; 2267: 57-71, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33786784

RESUMO

DNA replication is a fundamental process of life. Any perturbation of this process by endogenous or exogenous factors impacts on genomic stability and thereby on carcinogenesis. More recently, the replication machinery has been discovered as an interesting target for cancer therapeutic strategies. Given its high biological and clinical relevance, technologies for the analysis of DNA replication have attracted major attention. The so-called DNA fiber spreading technique is a powerful tool to directly monitor various aspects of the replication process by sequential incorporation of halogenated nucleotide analogs which later can be fluorescently stained and analyzed. This chapter outlines the use of the DNA fiber spreading technique for the analysis of replication dynamics and replication structures.


Assuntos
Replicação do DNA , Imagem Individual de Molécula/métodos , Animais , Linhagem Celular , DNA/química , DNA/genética , DNA/ultraestrutura , DNA Helicases/metabolismo , Halogênios/química , Humanos , Timidina/análogos & derivados
13.
Phys Chem Chem Phys ; 23(9): 5447-5454, 2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33650609

RESUMO

The population and depopulation mechanisms leading to the lowest-lying triplet states of 2-Se-Thymine were studied at the MS-CASPT2/cc-pVDZ level of theory. Several critical points on different potential energy hypersurfaces were optimized, including minima, conical intersections, and singlet-triplet crossings. The accessibility of all relevant regions on the potential energy hypersurfaces was investigated by means of minimum energy paths and linear interpolation in internal coordinates techniques. Our analysis indicates that, after the population of the bright S2 state in the Franck-Condon region, the first photochemical event is a barrierless evolution towards one of its two minima. After that, three viable photophysical deactivation paths can take place. In one of them, the population in the S2 state is transferred to the T2 state via intersystem crossing and subsequently to the T1 state by internal conversion. Alternatively, the S1 state could be accessed by internal conversion through two distinct conical intersections with S2 state followed by singlet-triplet crossing with the T2 state. The absence of a second minimum on the T1 state and a small energy barrier on pathway along the potential energy surface towards the ground state from the lowest triplet state are attributed as potential reasons to explain why the lifetime of the triplet state of 2-Se-Thymine might be reduced in comparison with its thio-analogue.


Assuntos
Compostos Organosselênicos/química , Timidina/análogos & derivados , Cinética , Modelos Moleculares , Conformação Molecular , Oxirredução , Processos Fotoquímicos , Termodinâmica , Timidina/química
14.
Nat Protoc ; 16(4): 1995-2022, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33627842

RESUMO

Quantification of cellular proliferation in humans is important for understanding biology and responses to injury and disease. However, existing methods require administration of tracers that cannot be ethically administered in humans. We present a protocol for the direct quantification of cellular proliferation in human hearts. The protocol involves administration of non-radioactive, non-toxic stable isotope 15Nitrogen-enriched thymidine (15N-thymidine), which is incorporated into DNA during S-phase, in infants with tetralogy of Fallot, a common form of congenital heart disease. Infants with tetralogy of Fallot undergo surgical repair, which requires the removal of pieces of myocardium that would otherwise be discarded. This protocol allows for the quantification of cardiomyocyte proliferation in this discarded tissue. We quantitatively analyzed the incorporation of 15N-thymidine with multi-isotope imaging spectrometry (MIMS) at a sub-nuclear resolution, which we combined with correlative confocal microscopy to quantify formation of binucleated cardiomyocytes and cardiomyocytes with polyploid nuclei. The entire protocol spans 3-8 months, which is dependent on the timing of surgical repair, and 3-4.5 researcher days. This protocol could be adapted to study cellular proliferation in a variety of human tissues.


Assuntos
Divisão Celular , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Miócitos Cardíacos/citologia , Timidina/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células , Feminino , Feto/citologia , Humanos , Imageamento Tridimensional , Lactente , Leucócitos/citologia , Miocárdio/citologia , Isótopos de Nitrogênio/urina , Ploidias , Gravidez , Sarcômeros/metabolismo , Tetralogia de Fallot/patologia
15.
Aging Cell ; 20(3): e13321, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33626245

RESUMO

One of the hallmarks of aging is an accumulation of cells with defects in oxidative phosphorylation (OXPHOS) due to mutations of mitochondrial DNA (mtDNA). Rapidly dividing tissues maintained by stem cells, such as the colonic epithelium, are particularly susceptible to accumulation of OXPHOS defects over time; however, the effects on the stem cells are unknown. We have crossed a mouse model in which intestinal stem cells are labelled with EGFP (Lgr5-EGFP-IRES-creERT2) with a model of accelerated mtDNA mutagenesis (PolgAmut/mut ) to investigate the effect of OXPHOS dysfunction on colonic stem cell proliferation. We show that a reduction in complex I protein levels is associated with an increased rate of stem cell cycle re-entry. These changes in stem cell homeostasis could have significant implications for age-associated intestinal pathogenesis.


Assuntos
Envelhecimento/patologia , Colo/patologia , Complexo I de Transporte de Elétrons/deficiência , Doenças Mitocondriais/patologia , Células-Tronco/patologia , Animais , Proliferação de Células , Feminino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Timidina/metabolismo
16.
Sci Rep ; 11(1): 1360, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33446731

RESUMO

Despite the tremendous advancements made in cell tracking, in vivo imaging and volumetric analysis, it remains difficult to accurately quantify the number of infused cells following stem cell therapy, especially at the single cell level, mainly due to the sensitivity of cells. In this study, we demonstrate the utility of both liquid scintillator counter (LSC) and accelerator mass spectrometry (AMS) in investigating the distribution and quantification of radioisotope labeled adipocyte derived mesenchymal stem cells (AD-MSCs) at the single cell level after intravenous (IV) transplantation. We first show the incorporation of 14C-thymidine (5 nCi/ml, 24.2 ng/ml) into AD-MSCs without affecting key biological characteristics. These cells were then utilized to track and quantify the distribution of AD-MSCs delivered through the tail vein by AMS, revealing the number of AD-MSCs existing within different organs per mg and per organ at different time points. Notably, the results show that this highly sensitive approach can quantify one cell per mg which effectively means that AD-MSCs can be detected in various tissues at the single cell level. While the significance of these cells is yet to be elucidated, we show that it is possible to accurately depict the pattern of distribution and quantify AD-MSCs in living tissue. This approach can serve to incrementally build profiles of biodistribution for stem cells such as MSCs which is essential for both research and therapeutic purposes.


Assuntos
Radioisótopos de Carbono , Rastreamento de Células , Espectrometria de Massas , Células-Tronco Mesenquimais/metabolismo , Compostos Radiofarmacêuticos , Timidina , Animais , Radioisótopos de Carbono/farmacocinética , Radioisótopos de Carbono/farmacologia , Xenoenxertos , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Timidina/farmacocinética , Timidina/farmacologia
17.
J Mass Spectrom ; 56(1): e4677, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33448524

RESUMO

The kinetics of the unimolecular dissociations of proton-bound dimers produced by fast-atom bombardment from nucleosides and reference amines enables the evaluation of the proton affinities (PAs) of ribonucleosides. The PAs of cytosine, guanosine, adenosine, uridine, and deoxyuridine have been thus determined. These values and those already available for the corresponding DNA homologues allow the evaluation of the effect of the hydroxyl group in position 2' of the sugar moiety, which lowers the PAs of RNA nucleosides by 0.6-1 kcal/mol, and of the methyl group in position 5 of the thymine ring, which enhances the basicity of deoxythymidine over deoxyuridine by 0.6 kcal/mol.


Assuntos
Nucleosídeos/química , Espectrometria de Massas por Ionização por Electrospray/métodos , DNA/química , Desoxiuridina/química , Cinética , Prótons , RNA/química , Timidina/química
18.
Ann Nucl Med ; 35(2): 211-222, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33387282

RESUMO

OBJECTIVES: The aim of this study was to assess the clinical value of [11C]4DST uptake in patients with lung nodules, including benign and malignant tumors, and to assess the correlation between [11C]4DST uptake and proliferative activity of tumors in comparison with [18F]FDG uptake. METHODS: Twenty-six patients (22 males and 4 females, mean age of 65.5-year-old) were analyzed in this prospective study. Patients underwent [11C]4DST and [18F]FDG PET/CT imaging on the same day. Diagnosis of each lung nodule was confirmed by histopathological examination of tissue specimens at surgery, or during clinical follow-up after the PET/CT studies. To assess the utility of the semi-quantitative evaluation method, the SUVmax was calculated of [11C]4DST and [18F]FDG uptake by the lesion. Proliferative activities of each tumor as indicated by the immunohistochemical Ki-67 index was also estimated using surgical specimens of patients. Then the relationship between the SUVmax of both PET/CT and the Ki-67 index was examined. Furthermore, the relationship between the uptake of [11C]4DST or [18F]FDG and the histopathological findings, the clinical stage, and the clinical outcome of patients were also assessed. RESULTS: There was a positive linear relationship between the SUVmax of [11C]4DST images and the Ki-67 index (Correlation coefficients = 0.68). The SUVmax of [11C]4DST in the 26 lung nodules were 1.65 ± 0.40 for benign lesions, 3.09 ± 0.83 for adenocarcinomas (P < 0.001 between benign and adenocarcinoma), and 2.92 ± 0.58 for SqCCs (P < 0.001 between benign and SqCC). Whereas, the SUVmax of [18F]FDG were 2.38 ± 2.27 for benign lesions, 6.63 ± 4.24 for adenocarcinomas (n.s.), and 7.52 ± 2.84 for SqCCs (n.s.). The relationship between TNM tumor stage and the SUVmax of [11C]4DST were 2.54 ± 0.37 for T1, 3.48 ± 0.57 for T2, and 4.17 ± 0.72 for T3 (P < 0.005 between T1 and T2, and P < 0.001 between T1 and T3). In comparison with the TNM pathological stage, SUVmax of [11C]4DST were 2.63 ± 0.49 for stage I, 3.36 ± 0.23 for stage II, 3.40 ± 1.12 for stage III, and 4.65 for stage IV (P < 0.05 between stages I and II). In comparison of the clinical outcome, the SUVmax of [11C]4DST were 2.72 ± 0.56 for the no recurrence (No Rec.) group, 3.10 ± 0.33 for the recurrence-free with adjuvant chemotherapy after the surgery (the No Rec. Adjv. CTx. group) and 4.66 ± 0.02 for the recurrence group (Rec. group) (P < 0.001 between the No Rec and Rec. groups, and P < 0.005 between the No Rec. Adjv. CTx. and Rec. groups). CONCLUSIONS: PET/CT with [11C]4DST is as feasible for imaging of lung tumors as [18F]FDG PET/CT. For diagnosing lung tumors, [11C]4DST PET is useful in distinguishing benign nodules from malignancies. [11C]4DST uptake in lung carcinomas is correlated with the proliferative activity of tumors, indicating a promising noninvasive PET imaging of DNA synthesis in malignant lung tumors.


Assuntos
Radioisótopos de Carbono/química , Radioisótopos de Flúor/química , Neoplasias Pulmonares/diagnóstico por imagem , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Compostos Radiofarmacêuticos/química , Timidina/química , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/classificação , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estudos Prospectivos
19.
Bioorg Med Chem ; 31: 115966, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33387694

RESUMO

Thymidine derivatives bearing spiroacetal moieties on the C4'-position (5'R-spiro-thymidine and 5'S-spiro-thymidine) were synthesized and incorporated into oligonucleotides. The duplex- and triplex-forming abilities of both the oligonucleotides were evaluated from UV melting experiments. Oligonucleotides with the 5'S-spiro modifications could form thermally stable duplexes with complementary RNA and DNA; however, the 5'R-spiro modification significantly decreased the thermal stabilities of the duplexes and triplexes. Oligonucleotides with these spiro-thymidines showed significantly high resistance towards enzymatic degradation.


Assuntos
Oligonucleotídeos/química , Compostos de Espiro/química , Timidina/química , Estrutura Molecular , Oligonucleotídeos/síntese química
20.
Org Biomol Chem ; 19(6): 1292-1295, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33508059

RESUMO

We describe highly efficient interstrand photocrosslinking of a DNA duplex containing 5-chloro-2'-deoxy-4-thiouridine (ClSdU) in one strand, proceeding via a two-step photochemical cascade, involving the formation of a thermally reversible crosslink between ClSdU and thymidine in the target strand and its subsequent conversion to a thermally stable fluorescent crosslink. These results show that ClSdU has great potential to be a valuable DNA photo-crosslinking reagent for chemical biology applications.


Assuntos
Reagentes para Ligações Cruzadas/química , DNA/química , Oligodesoxirribonucleotídeos/química , Tiouridina/análogos & derivados , Reagentes para Ligações Cruzadas/efeitos da radiação , Temperatura , Tiouridina/efeitos da radiação , Timidina/química , Raios Ultravioleta
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