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1.
Ann Lab Med ; 42(2): 196-202, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34635613

RESUMO

Background: Identifying the causal pathogen of encephalitis remains a clinical challenge. A 50-year-old man without a history of neurological disease was referred to our department for the evaluation of an intracranial lesion observed on brain magnetic resonance imaging (MRI) scans, and the pathology results suggested protozoal infection. We identified the species responsible for encephalitis using thymine-adenine (TA) cloning, suitable for routine clinical practice. Methods: We extracted DNA from a paraffin-embedded brain biopsy sample and performed TA cloning using two universal eukaryotic primers targeting the V4-5 and V9 regions of the 18S rRNA gene. The recombinant plasmids were extracted, and the inserted amplicons were identified by Sanger sequencing and a homology search of sequences in the National Center for Biotechnology Information Basic Local Alignment Search Tool. Results: The infection was confirmed to be caused by the free-living amoeba Balamuthia mandrillaris. Two of 41 colonies recombinant with 18S V4-5 primers and 35 of 63 colonies recombinant with the 18S V9 primer contained B. mandrillaris genes; all other colonies contained human genes. Pathogen-specific PCR ruled out Entamoeba histolytica, Naegleria fowleri, Acanthamoeba spp., and Toxoplasma gondii infections. Conclusions: This is the first report of B. mandrillaris-induced encephalitis in Korea based on molecular identification. TA cloning with the 18S rRNA gene is a feasible and affordable diagnostic tool for the detection of infectious agents of unknown etiology.


Assuntos
Balamuthia mandrillaris , Encefalite , Adenina , Balamuthia mandrillaris/genética , Clonagem Molecular , Encefalite/diagnóstico , Eucariotos , Humanos , Masculino , Pessoa de Meia-Idade , Timina
2.
Talanta ; 238(Pt 1): 123009, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34857343

RESUMO

A new fluorescent sensor is introduced to analyze nucleoside analogue, trifluridine and tipiracil in tablets and biological fluids. The synthesized fluorophore exhibits good fluorescence at 446 nm after excitation at 257 nm. The interaction between the studied drugs and the reagent was a quenching effect. Different experimental parameters and the mechanism of quenching were discussed. The present method was utilized to analyze trifluridine and tipiracil raw materials and tablets over the concentration range of 20-1000 ng/mL and spiked biological fluids over the range of 30-1000 ng/mL. The method is selective, specific, and possesses good accuracy and high precision. The method is highly sensitive, with detection limits of 5.8 and 6.0 ng/mL for trifluridine and tipiracil, respectively, and quantitation limits of 17.7 and 18.1 ng/mL for trifluridine and tipiracil, respectively. In vivo analysis of trifluridine was achieved selectively and the mean pharmacokinetic parameters were studied.


Assuntos
Neoplasias Colorretais , Trifluridina , Protocolos de Quimioterapia Combinada Antineoplásica , Combinação de Medicamentos , Humanos , Nucleosídeos , Pirrolidinas , Timina , Trifluridina/uso terapêutico , Uracila/uso terapêutico
3.
Phys Chem Chem Phys ; 24(2): 829-841, 2022 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-34928284

RESUMO

Hexagonal boron nitride (h-BN) sheets possess high fluorescence quenching ability and high affinity towards DNA/RNA, and they can be used as a sensing platform for rapid detection. We report the absorption and emission properties of DNA nucleobases such as adenine (A), cytosine (C), guanine (G), and thymine (T) tagged with benzoxazole on h-BN and aluminium-doped h-BN (Al_hBN) sheets. The binding affinity of studied nucleobases on h-BN sheets at the M062X/6-31G* level of theory showed the following adsorption trend: G ≥ T ≥ A > C, which is in good agreement with the previous results. The calculated stability trend of nucleobases on the Al_hBN sheet follows as C > G > A > T at the same level of theory. The physically adsorbed behavior of nucleobases to h-BN sheets was confirmed by the non-covalent interactions (NCIs) and the total density of states (TDOS) plots. The NCI results indicated that van der Waals interactions contribute significantly to the adsorption of nucleobases on h-BN sheets. Atoms in molecules (AIM) calculations revealed the electrostatic interactions between nucleobases and the Al_hBN sheet. The quenching phenomenon of nucleobase-tagged fluorophores on h-BN and Al_hBN sheets was investigated by TD-DFT calculations using the same level of theory. The thymine-tagged fluorophore upon adsorption to the pristine h-BN sheet was found to be blue-shifted (∼43 nm); however, the guanine-tagged fluorophore with Al_hBN showed a remarkable difference from other nucleobase-tagged fluorophores in the absorption and emission spectrum. Guanine-tagged fluorophores showed a smaller blue shift (∼7 nm) in the absorption spectrum; however, it showed a larger red shift (∼55 nm) than the other nucleobase-tagged fluorophores on Al_hBN sheets and can be useful in recognizing a sequence-specific phenomenon as a fluorescent biosensor of DNA and RNA to ascertain the presence of such nucleobases.


Assuntos
Alumínio/química , Benzoxazóis/química , Compostos de Boro/química , Teoria da Densidade Funcional , Corantes Fluorescentes/química , Simulação de Dinâmica Molecular , Adenina/química , Adsorção , Citosina/química , Guanina/química , Timina/química
4.
Phys Chem Chem Phys ; 23(40): 23005-23013, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34611693

RESUMO

Core ionization of DNA begins a cascade of events which could lead to cellular inactivation or death. The created core-hole following an impulse inner-shell ionization of molecules naturally decays in the auger timescale. We simulated charge migration (CM) phenomena following an impulsive core ionization of individual DNA bases at the oxygen K-edge which occurs before Auger decay of the oxygen. Our approach is based on real-time time dependent density functional theory (RT-TDDFT). It is shown that the pronounced hole fluctuation observed around bonds of the initial core-hole results in various valence orbital migrations. Also, the same photo-core-ionized dynamics is studied for the related base pairs. We investigate the role of base pairing and H-bonding interactions in the attosecond CM dynamics. In particular, the creation of a core-hole in the oxygen involved in H-bonding leads to an enhancement of charge migration relative to the respective single bases. Importantly, the hole oscillation of the adenine-thymine base pair upon creation of a core-hole at the oxygen, which does not contribute to the donor-acceptor interactions (not H-bonded), decreases compared to the single thymine base. Understanding the detailed dynamics of the localized core-hole initiating CM process would open the way for chemically controlling DNA damage/repair in the future.


Assuntos
DNA/química , Oxigênio/química , Adenina/química , Adenina/metabolismo , Pareamento de Bases , DNA/metabolismo , Teoria da Densidade Funcional , Ligação de Hidrogênio , Íons/química , Timina/química , Timina/metabolismo
5.
Biophys J ; 120(20): 4325-4336, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34509507

RESUMO

Repeat-induced point mutation is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. Repeat-induced point mutation detects duplications (irrespective of their origin, specific sequence, coding capacity, and genomic positions) by a recombination-independent mechanism that likely matches intact DNA double helices directly, without relying on the annealing of complementary single strands. In the fungus Neurospora crassa, closely positioned repeats can induce mutation of the adjoining nonrepetitive regions. This process is related to heterochromatin assembly and requires the cytosine methyltransferase DIM-2. Using DIM-2-dependent mutation as a readout of homologous pairing, we find that GC-rich repeats produce a much stronger response than AT-rich repeats, independently of their intrinsic propensity to become mutated. We also report that direct repeats trigger much stronger DIM-2-dependent mutation than inverted repeats. These results can be rationalized in the light of a recently proposed model of homologous DNA pairing, in which DNA double helices associate by forming sequence-specific quadruplex-based contacts with a concomitant release of supercoiling. A similar process featuring pairing-induced supercoiling may initiate epigenetic silencing of repetitive DNA in other organisms, including humans.


Assuntos
Citosina , DNA Fúngico , Recombinação Genética , Timina , DNA Fúngico/genética , Mutação , Neurospora crassa/genética
6.
ESMO Open ; 6(5): 100270, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34547581

RESUMO

BACKGROUND: In preclinical studies trifluridine/tipiracil (FTD/TPI) plus oxaliplatin (Industriestrasse, Holzkirchen, Germany) sensitised microsatellite stable (MSS) metastatic colorectal cancer (mCRC) to anti-programmed cell death protein-1; the addition of oxaliplatin or bevacizumab (F Hoffmann- la ROCHE AG, Kaiseraugst, Switzerland) enhanced the antitumour effects of FTD/TPI. This study aimed to investigate the safety and efficacy of FTD/TPI plus oxaliplatin and either bevacizumab or nivolumab (Uxbridge business Park, Uxbridge, United Kingdom) in patients with mCRC who had progressed after at least one prior line of treatment. PATIENTS AND METHODS: In 14-day cycles, patients received FTD/TPI 35 mg/m2 (twice daily, days 1-5) plus oxaliplatin 85 mg/m2 (day 1), and, on day 1, either bevacizumab 5 mg/kg (cohort A) or nivolumab 3 mg/kg (cohort B). Patients in Cohort B had confirmed MSS status. RESULTS: In total, 54 patients were enrolled: 37 in cohort A and 17 in cohort B. Recruitment in cohort B was stopped early due to the low response rate (RR) observed at interim analyses of efficacy. The most common adverse events (AEs) in cohort A were neutropenia/decreased neutrophils (75.7%), nausea (59.5%), vomiting (40.5%), diarrhoea (37.8%), peripheral sensory neuropathy (37.8%), fatigue (35.1%) and decreased appetite (35.1%). In cohort B, the most common AEs were neutropenia/decreased neutrophils (70.6%), diarrhoea (58.8%), nausea (47.1%), vomiting (47.1%), fatigue (47.1%), asthenia (41.2%), paraesthesia (41.2%), thrombocytopenia/decreased platelets (35.3%) and decreased appetite (35.3%). Confirmed objective RR was 17.1% in cohort A and 7.1% in cohort B; the corresponding values for median progression-free survival in the two cohorts were 6.3 and 6.0 months. CONCLUSION: FTD/TPI plus oxaliplatin and bevacizumab or nivolumab had an acceptable safety profile and demonstrated antitumour activity in previously treated patients with mCRC.


Assuntos
Neoplasias Colorretais , Trifluridina , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bevacizumab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Humanos , Nivolumabe/uso terapêutico , Oxaliplatina/uso terapêutico , Pirrolidinas , Timina , Trifluridina/uso terapêutico
7.
J Phys Chem A ; 125(37): 8196-8204, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34516113

RESUMO

According to the Löwdin model [ Rev. Mod. Phys. 1963, 35, 724-732], the Watson-Crick guanine-cytosine (G-C) base pair is tautomerized (G*-C*) with a small probability and then replication of G*-C* produces G*-thymine (T) and adenine (A)-C* base pairs. On the basis of this model and our previous work [ J. Phys. Chem. B 2020, 124, 1715-1722], we first calculated the intrinsic reaction coordinates from G*-T to G-T* using density functional theory and evaluated the probability of G*-T tautomerization to G-T* by double proton transfer (DPT) on the basis of the transition state theory. Similarly, we calculated the probability of A-C* tautomerization to A*-C by DPT. Then, according to these probabilities, we calculated the probability of transition mutations from G-C to A-T after 2 replications. The calculated probability was 1.31 × 10-8, a value consistent with the mutation rate previously reported by Drake et al. [ Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 7160-7164]. Our results suggest that DPT is one cause of the G-C → A-T transition. To investigate differences in the optical properties between G*-T and G-T* and between A-C* and A*-C, we also evaluated the infrared absorption spectra and Raman intensities for these base pairs.


Assuntos
Adenina/química , Citosina/química , Guanina/química , Prótons , Timina/química , Pareamento de Bases , Teoria da Densidade Funcional , Cinética
8.
Free Radic Biol Med ; 174: 321-328, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34339797

RESUMO

Pterin (Ptr) is a model photosensitizer that acts mainly through type I mechanism and is able to photoinduce the one-electron oxidation of purine and pyrimidine nucleobases. However, under anaerobic conditions Ptr reacts with thymine (T) to form photoadducts (Ptr-T) but does not lead to the photodegradation of guanine (G), which is the nucleobase with the lowest ionization potential. Accordingly, G is thermodynamically able to reduce the radicals of the other nucleobases and has been described in this sense as the "hole sink" of the DNA double helix. Here we analyze by steady-state and time-resolved studies the effect of G in the anaerobic photosensitization of T by Ptr, using nucleotides and oligonucleotides of different sequences. We demonstrated that G is able to reduce T radicals but does not prevent the formation of Ptr-T adducts. Our results suggest that after the encounter between the excited Ptr and T, and completion of the electron transfer step, part of the radicals escape from the solvent cage, to further react with other species. However, a proportion of radicals do not escape and evolve to photoadducts before separation. We provide new evidence that contributes to understand the photosensitizing properties of Ptr in the absence of O2, the mechanism of formation of photoadducts in the DNA and the protective role of G towards the photodamage in other nucleobases.


Assuntos
Pterinas , Timina , Anaerobiose , Guanina , Oxirredução
9.
Nucleic Acids Res ; 49(15): 8923-8933, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34289059

RESUMO

The most common form of DNA methylation involves the addition of a methyl group to a cytosine base in the context of a cytosine-phosphate-guanine (CpG) dinucleotide. Genomes from more primitive organisms are more abundant in CpG sites that, through the process of methylation, deamination and subsequent mutation to thymine-phosphate-guanine (TpG) sites, can produce new transcription factor binding sites. Here, we examined the evolutionary history of the over 36 000 glucocorticoid receptor (GR) consensus binding motifs in the human genome and identified a subset of them in regulatory regions that arose via a deamination and subsequent mutation event. GR can bind to both unmodified and methylated pre-GR binding sequences (GBSs) that contain a CpG site. Our structural analyses show that CpG methylation in a pre-GBS generates a favorable interaction with Arg447 mimicking that made with a TpG in a GBS. This methyl-specific recognition arose 420 million years ago and was conserved during the evolution of GR and likely helps fix the methylation on the relevant cytosines. Our study provides the first genetic, biochemical and structural evidence of high-affinity binding for the likely evolutionary precursor of extant TpG-containing GBS.


Assuntos
Metilação de DNA/genética , Evolução Molecular , Genoma Humano/genética , Receptores de Glucocorticoides/genética , Sítios de Ligação/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Fosfatos de Dinucleosídeos/genética , Humanos , Conformação de Ácido Nucleico , Receptores de Glucocorticoides/ultraestrutura , Sequências Reguladoras de Ácido Nucleico/genética , Timina/química
10.
Sci Rep ; 11(1): 14321, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253805

RESUMO

Trifluridine/tipiracil increases overall survival (OS) in patients with refractory, metastatic colorectal cancer (mCRC). A post hoc exploratory analysis of the RECOURSE randomized clinical trial (RCT) established two categories, a good prognosis corresponding to subjects having a low tumor burden and indolent disease. Other models in refractory mCRC are the FAS-CORRECT and Colon Life nomogram. The main objective was to externally validate the prognostic factors of the RECOURSE and FAS-CORRECT trials, and the Colon Life nomogram in a multicenter, real-world series of mCRC treated in 3rd and successive lines with trifluridine/tipiracil. The secondary aim was to develop an OS predictive model, TAS-RECOSMO. Between 2016 and 2019, 244 patients were recruited. Median OS was 8.15 vs 8.12 months for the poor (85% of the subjects) and good (15%) prognosis groups from the RESOURCE trial, respectively, log-rank p = 0.9. The most common grade 3-4 toxicities were neutropenia (17%), asthenia (6%), and anemia (5%). The AFT lognormal model TAS-RECOSMO included six variables: ECOG-PS, KRAS/NRAS/BRAF mutation status, time between diagnosis of metastasis and beginning of trifluridine/tipiracil, NLR, CEA, and alkaline phosphatase. The model's bootstrapped bias-corrected c-index was 0.682 (95% CI, 0.636-0.722). The factors from the Colon Life model, FAS-CORRECT, and RECOURSE displayed a c-index of 0.690, 0.630, and 0.507, respectively. TAS-RECOSMO, FAS-CORRECT, and the Colon Life nomogram appear to predict OS in patients with refractory mCCR who begin trifluridine/tipiracil treatment in the real world. The prognostic groups of the RECOURCE RCT were unable to capture the situation of real-world subjects treated with trifluridine/tipiracil in this series.


Assuntos
Neoplasias Colorretais/tratamento farmacológico , Pirrolidinas/uso terapêutico , Timina/uso terapêutico , Trifluridina/uso terapêutico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Astenia/tratamento farmacológico , Combinação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Multicêntricos como Assunto , Neutropenia/tratamento farmacológico , Adulto Jovem
11.
Commun Biol ; 4(1): 862, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253820

RESUMO

Breast carcinomas commonly carry mutations in the tumor suppressor p53, although therapeutic efforts to target mutant p53 have previously been unfruitful. Here we report a selective combination therapy strategy for treatment of p53 mutant cancers. Genomic data revealed that p53 mutant cancers exhibit high replication activity and express high levels of the Base-Excision Repair (BER) pathway, whereas experimental testing showed substantial dysregulation in BER. This defect rendered accumulation of DNA damage in p53 mutant cells upon treatment with deoxyuridine analogues. Notably, inhibition of poly (ADP-ribose) polymerase (PARP) greatly enhanced this response, whereas normal cells responded with activation of the p53-p21 axis and cell cycle arrest. Inactivation of either p53 or p21/CDKN1A conferred the p53 mutant phenotype. Preclinical animal studies demonstrated a greater anti-neoplastic efficacy of the drug combination (deoxyuridine analogue and PARP inhibitor) than either drug alone. This work illustrates a selective combination therapy strategy for p53 mutant cancers that will improve survival rates and outcomes for thousands of breast cancer patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Reparo do DNA/genética , Mutação , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Células A549 , Animais , Linhagem Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Combinação de Medicamentos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Camundongos SCID , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Pirrolidinas/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Timina/administração & dosagem , Trifluridina/administração & dosagem , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Proteína Supressora de Tumor p53/metabolismo
12.
Anal Chem ; 93(30): 10495-10501, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34293865

RESUMO

Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.


Assuntos
Azidas , Timina , Química Click , DNA , Estresse Oxidativo
13.
Curr Oncol ; 28(3): 2260-2269, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34207352

RESUMO

In the UK, Trifluridine-tipiracil (Lonsurf) is used to treat metastatic colorectal cancer in the third-line setting, after prior exposure to fluoropyrimidine-based regimes. Current data on the real-world use of Lonsurf lack long-term follow-up data. A retrospective evaluation of patients receiving Lonsurf at our Cancer Centre in 2016-2017 was performed, all with a minimum of two-year follow-up. Fifty-six patients were included in the review. The median number of cycles of Lonsurf administered was 3. Median follow-up was 6.0 months, with all patients deceased at the time of analysis. Median progression-free survival (PFS) was 3.2 months, and overall survival (OS) was 5.8 months. The median interval from Lonsurf discontinuation to death was two months, but seven patients received further systemic treatment and median OS gained was 12 months. Lonsurf offered a slightly better PFS but inferior OS to that of the RECOURSE trial, with PFS similar to real-world data previously presented. Interestingly, 12.5% had a PFS > 9 months, and this cohort had primarily left-sided and RAS wild-type disease. A subset received further systemic treatment on Lonsurf discontinuation with good additional OS benefit. Lonsurf may alter the course of disease for a subset of patients, and further treatment on progression can be considered in carefully selected patients.


Assuntos
Neoplasias Colorretais , Trifluridina , Neoplasias Colorretais/tratamento farmacológico , Combinação de Medicamentos , Humanos , Pirrolidinas , Estudos Retrospectivos , Timina , Reino Unido
14.
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
15.
J Phys Chem B ; 125(25): 6889-6896, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34137627

RESUMO

Hydroxychloroquine (HCQ) is an important drug for the treatment of rheumatoid arthritis and malaria. HCQ targets specifically to nucleic acids for its action. However, the mechanism of HCQ binding and the effect of its binding on the stability of DNA are elusive. In this study, the binding mechanism of HCQ and the effect of binding on stability of different sequences of DNA have been investigated using spectroscopic and molecular dynamics (MD) simulation techniques. HCQ binds with all of the sequences of DNA and stabilizes them. However, binding efficacy of HCQ with DNA depends on its sequences as the binding constant is highest for pure guanine-cytosine (G-C) rich DNA and decreases with the increase of adenine-thymine (A-T) bases. HCQ prefers to interact with AT DNA through the minor groove whereas the major groove along with intercalation are the favorable binding mode in the case of GC DNA. The binding of HCQ in the major groove of GC DNA enhances the stacking between the bases compared to the case of AT DNA which leads to higher stability for GC DNA. It appears that the groove switching of HCQ is correlated with binding affinity as well as stability of different sequences of DNA.


Assuntos
DNA , Hidroxicloroquina , Citosina , Simulação de Dinâmica Molecular , Timina
16.
Anticancer Res ; 41(6): 3131-3137, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34083307

RESUMO

BACKGROUND/AIM: Our multicenter phase II TAS-CC3 study demonstrated favorable median progression-free survival (PFS) and overall survival (OS) of 32 metastatic colorectal cancer (mCRC) patients treated with TAS-102 + bevacizumab as 3rd-line treatment. PATIENTS AND METHODS: We investigated the predictive and prognostic values of pre-treatment blood inflammation-based scores, including the neutrophil-to-lymphocyte (NLR), platelet-to-lymphocyte (PLR) and lymphocyte-to-monocyte ratio (LMR) on disease-control (DC), PFS and OS by a post-hoc analysis. RESULTS: Receiver operating characteristic curve analyses of the 3 inflammation-based scores versus DC showed the best predictive performance for LMR, followed by NLR and PLR. The high-LMR group had a significantly higher DC rate than the low group (87.5 vs. 43.8%). The high-LMR group showed significantly longer survival than the low group (4.9 vs. 2.3 m for median PFS) (21.0 vs. 6.1 m for median OS). CONCLUSION: The pre-treatment LMR is a valid predictive and prognostic biomarker for mCRC patients undergoing TAS-102 and bevacizumab treatment.


Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Bevacizumab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Linfócitos/patologia , Monócitos/patologia , Metástase Neoplásica/tratamento farmacológico , Pirrolidinas/uso terapêutico , Timina/uso terapêutico , Trifluridina/uso terapêutico , Idoso , Antineoplásicos Imunológicos/administração & dosagem , Bevacizumab/administração & dosagem , Neoplasias Colorretais/patologia , Combinação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pirrolidinas/administração & dosagem , Timina/administração & dosagem , Trifluridina/administração & dosagem
17.
Cancer Chemother Pharmacol ; 88(3): 485-497, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34097100

RESUMO

PURPOSE: Trifluridine/tipiracil (FTD/TPI) is approved for advanced colorectal and gastric/gastroesophageal cancer; however, data in patients with renal impairment (RI) are limited. This phase I study evaluated FTD/TPI in patients with advanced solid tumors and varying degrees of RI to develop dosing guidance. METHODS: Patients were enrolled into normal renal function (CrCl ≥ 90 mL/min), mild RI (CrCl 60-89 mL/min), or moderate RI (CrCl 30-59 mL/min) cohorts and administered the recommended FTD/TPI dose (35 mg/m2 twice daily, days 1-5 and 8-12; 28-day cycle). Based on interim pharmacokinetics/safety data, patients with severe RI (CrCl 15-29 mL/min) were enrolled and received FTD/TPI 20 mg/m2 twice daily. RESULTS: Forty-three patients (normal renal function [n = 12]; mild RI [n = 12]; moderate RI [n = 11]; severe RI [n = 8]) were enrolled and treated. At steady state, compared to values in patients with normal renal function, FTD area under the curve (AUC) was not significantly different in patients with RI, but TPI AUC was significantly higher and increased with RI severity. FTD/TPI safety profile was consistent with prior experience, but grade ≥ 3 adverse events (AEs) were more frequent in the RI cohorts (83.3% [mild], 90.9% [moderate], 75.0% [severe], and normal [50.0%]). Hematologic AEs (anemia and neutropenia) were more frequent with RI. Overall, seven patients discontinued because of unrelated, nonhematologic AEs. CONCLUSION: FTD/TPI is safe and tolerable at the recommended 35 mg/m2 dose in patients with mild/moderate RI and at the reduced 20 mg/m2 dose in patients with severe RI. TRIAL REGISTRATION: NCT02301117, registration date: November 21, 2014.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Nefropatias/fisiopatologia , Neoplasias/tratamento farmacológico , Pirrolidinas/administração & dosagem , Timina/administração & dosagem , Trifluridina/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia/induzido quimicamente , Anemia/epidemiologia , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Área Sob a Curva , Estudos de Coortes , Combinação de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neutropenia/induzido quimicamente , Neutropenia/epidemiologia , Pirrolidinas/efeitos adversos , Pirrolidinas/farmacocinética , Índice de Gravidade de Doença , Timina/efeitos adversos , Timina/farmacocinética , Trifluridina/efeitos adversos , Trifluridina/farmacocinética
18.
Jpn J Clin Oncol ; 51(9): 1383-1390, 2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34128042

RESUMO

OBJECTIVE: Nivolumab and trifluridine/tipiracil have significantly improved the overall survival of patients with heavily pretreated metastatic gastric cancer in different placebo-controlled phase III trials. Accordingly, nivolumab and trifluridine/tipiracil have been approved and recommended for patients with heavily pretreated metastatic gastric cancer in Japan. The aim of this study was to assess the cost-effectiveness of trifluridine/tipiracil against nivolumab. METHODS: A partitioned survival model, which consisted of three health states, namely, 'pre-progression,' 'post-progression,' and 'death,' was constructed. Efficacy and safety data were derived from the TAGS and ATTRACTION-2 trials. Costs were estimated based on the standard clinical pathway and national insurance fee schedules. One-way and probabilistic sensitivity analyses were performed. The threshold value was set to JPY 7 500 000 (USD 68 182) for each quality-adjusted life-year. RESULTS: The expected median overall survival and progression-free survival were 5.59 and 1.99 months for trifluridine/tipiracil and 5.26 and 1.55 months for nivolumab, respectively. The quality-adjusted life-year and expected costs per patient were 0.4379 and JPY 2 054 625 (USD 18 678) for trifluridine/tipiracil and 0.5295 and JPY 5 018 148 (USD 45 620) for nivolumab, respectively. The expected median progression-free survival and overall survival were equivalent with trifluridine/tipiracil and nivolumab, whereas the expected quality-adjusted life-year with trifluridine/tipiracil was slightly lower than that with nivolumab. However, trifluridine/tipiracil reduced the total treatment cost by JPY 2 963 523 (USD 26 996) compared with that of nivolumab. The incremental cost-effectiveness ratio of nivolumab versus trifluridine/tipiracil was JPY 32 352 489 (USD 294 113) per quality-adjusted life-year gained. CONCLUSIONS: Trifluridine/tipiracil was more cost-effective than nivolumab for patients with heavily pretreated metastatic gastric cancer.


Assuntos
Neoplasias Colorretais , Neoplasias Gástricas , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Análise Custo-Benefício , Combinação de Medicamentos , Humanos , Japão , Nivolumabe/uso terapêutico , Pirrolidinas , Neoplasias Gástricas/tratamento farmacológico , Timina , Trifluridina/uso terapêutico , Uracila/uso terapêutico
19.
ESMO Open ; 6(4): 100200, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34175675

RESUMO

BACKGROUND: Metastatic gastric cancer and cancer of the esophagogastric junction (GC/EGJ) is an aggressive disease with poor prognosis. In the TAGS study, trifluridine/tipiracil (FTD/TPI) improved overall survival (OS) compared with placebo in heavily pre-treated patients. This unplanned, exploratory subgroup analysis of the TAGS study aimed to clarify outcomes when FTD/TPI was used as third-line (3L) treatment and fourth- or later-line (4L+) treatment. PATIENTS AND METHODS: Patients were divided into a 3L group (126 and 64 in FTD/TPI and placebo arms, respectively) and 4L+ group (211 and 106 in FTD/TPI and placebo arms, respectively). Endpoints included OS, progression-free survival (PFS), time to Eastern Cooperative Oncology Group performance status (ECOG PS) deterioration to ≥2, and safety. RESULTS: Baseline characteristics were generally well balanced between FTD/TPI and placebo for 3L and 4L+ treatment. Median OS (mOS) for FTD/TPI versus placebo was: 6.8 versus 3.2 months {hazard ratio (HR) [95% confidence interval (CI)] = 0.68 (0.47-0.97), P = 0.0318} in the 3L group; and 5.2 versus 3.7 months [0.73 (0.55-0.95), P = 0.0192] in the 4L+ group. Median PFS for FTD/TPI versus placebo was 3.1 versus 1.9 months [0.54 (0.38-0.77), P = 0.0004] in the 3L group; and 1.9 versus 1.8 months [0.57 (0.44-0.74), P < 0.0001] in the 4L+ group. Time to deterioration of ECOG PS to ≥2 for FTD/TPI versus placebo was 4.8 versus 2.0 months [HR (95% CI) = 0.60 (0.42-0.86), P = 0.0049] in the 3L group; and 4.0 versus 2.5 months [0.75 (0.57-0.98), P = 0.0329] in the 4L+ group. The safety of FTD/TPI was consistent in all subgroups. CONCLUSIONS: This analysis confirms the efficacy and safety of FTD/TPI in patients with GC/EGJ in third and later lines with a survival benefit that seems slightly superior in 3L treatment. When FTD/TPI is taken in 3L as recommended in the international guidelines, physicians can expect to provide patients with an mOS of 6.8 months.


Assuntos
Neoplasias Colorretais , Neoplasias Gástricas , Humanos , Pirrolidinas , Neoplasias Gástricas/tratamento farmacológico , Timina , Trifluridina/uso terapêutico , Uracila
20.
Int J Mol Sci ; 22(10)2021 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-34064609

RESUMO

Monogenic hypertension is rare and caused by genetic mutations, but whether factors associated with mutations are disease-specific remains uncertain. Given two factors associated with high mutation rates, we tested how many previously known genes match with (i) proximity to telomeres or (ii) high adenine and thymine content in cardiovascular diseases (CVDs) related to vascular stiffening. We extracted genomic information using a genome data viewer. In human chromosomes, 64 of 79 genetic loci involving >25 rare mutations and single nucleotide polymorphisms satisfied (i) or (ii), resulting in an 81% matching rate. However, this high matching rate was no longer observed as we checked the two factors in genes associated with essential hypertension (EH), thoracic aortic aneurysm (TAA), and congenital heart disease (CHD), resulting in matching rates of 53%, 70%, and 75%, respectively. A matching of telomere proximity or high adenine and thymine content projects the list of loci involving rare mutations of monogenic hypertension better than those of other CVDs, likely due to adoption of rigorous criteria for true-positive signals. Our data suggest that the factor-disease matching rate is an accurate tool that can explain deleterious mutations of monogenic hypertension at a >80% match-unlike the relatively lower matching rates found in human genes of EH, TAA, CHD, and familial Parkinson's disease.


Assuntos
Doenças Cardiovasculares/genética , Predisposição Genética para Doença , Mutação , Doenças do Sistema Nervoso/genética , Polimorfismo de Nucleotídeo Único , Telômero/genética , Adenina/metabolismo , Doenças Cardiovasculares/metabolismo , Doenças Cardiovasculares/patologia , Humanos , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Timina/metabolismo
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