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1.
Ann Lab Med ; 42(1): 96-99, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34374354

RESUMO

The sensitivity of molecular diagnostics could be affected by nucleotide variants in pathogen genes, and the sites affected by such variants should be monitored. We report a single-nucleotide variant (SNV) in the nucleocapsid (N) gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), i.e., G29179T, which impairs the diagnostic sensitivity of the Xpert Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, CA, USA). We observed significant differences between the threshold cycle (Ct) values for envelope (E) and N genes and confirmed the SNV as the cause of the differences using Sanger sequencing. This SNV, G29179T, is the most prevalent in Korea and is associated with the B.1.497 virus lineage, which is dominant in Korea. Clinical laboratories should be aware of the various SNVs in the SARS-CoV-2 genome and consider their potential effects on the diagnosis of coronavirus disease 2019.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Técnicas de Diagnóstico Molecular , Nasofaringe , Nucleotídeos , Prevalência , República da Coreia , Sensibilidade e Especificidade
2.
Food Chem ; 370: 131352, 2022 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-34788963

RESUMO

Assessing the umami taste of seaweed on a chemical level can inform the use and selection of seaweed in European cuisine. Accordingly, we developed a method for the simultaneous extraction, separate clean-up and analysis of 21 free amino acids and 10 free nucleotides by reversed phase and mixed-mode HPLC respectively. Of multiple mouth emulating solvents, extracting in Milli-Q at 35 °C was found most suitable. This method showed good linearity (R2 > 0.9996), resolution (Rs ≥ 1.5) and picomole detection limits, and was successfully applied to determine the Equivalent Umami Concentration (EUC) and Taste Activity Values (TAV) of seven Dutch seaweed species. Phaeophyceae showed the highest EUC, followed by Chlorophyceae and Rhodophyceae (≈ 9.5, 3.7 and 1.1 g/100 g respectively). Glutamic acid always exceeded the TAV, while other umami compounds were species specific. Our method can accurately predict umami intensity and therefore contributes towards species selection for the European palette.


Assuntos
Nucleotídeos , Alga Marinha , Aminoácidos , Ácido Glutâmico , Paladar
3.
Zhongguo Yi Xue Ke Xue Yuan Xue Bao ; 43(5): 788-795, 2021 Oct.
Artigo em Chinês | MEDLINE | ID: mdl-34728041

RESUMO

Alzheimer's disease(AD)is a chronic neurodegenerative disease whose cause remains unclear.The ß-amyloid plaques in the brain are one of the major pathological features of AD.However,the drugs targeting extracellular ß-amyloid plaques have failed to cure the disease.Innate immunity and neuroinflammation play a role in the pathogenesis and progression of AD.As the macrophages existing in the central nervous system,microglia are related with extracellular ß-amyloid deposition,intracellular neurofibrillary tangle formation,and neuron injury.Accumulating evidence demonstrates that the activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome in microglia plays a role in AD,suggesting new therapeutic target for AD in this signaling pathway.This article reviewed the studies about the activation and regulation of NLRP3 inflammasome in the pathogenesis and progression of AD as well as the development of AD therapies targeting this pathway,aiming to provide reference for further studies in this field.


Assuntos
Doença de Alzheimer , Doenças Neurodegenerativas , Humanos , Inflamassomos , Microglia , Proteína 3 que Contém Domínio de Pirina da Família NLR , Nucleotídeos , Domínio Pirina
4.
Anal Chim Acta ; 1188: 339167, 2021 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-34794583

RESUMO

ATP-sensitive potassium (KATP) channels couple intracellular metabolism to the electrical activity by regulating K+ flux across the plasma membrane, thus playing an important role in both normal and pathophysiology. To understand the mechanism of ATP regulating biological ion channels, developing an ATP-responsive artificial nanochannel is an appealing but challenging topic because KATP channel is a heteromultimer of two subunits (potassium channel subunit (Kir6.x) and sulfonylurea receptor (SUR)) and exhibit dynamic functions with adjustability and reversibility. Inspired by the structure of KATP channels, we designed a smart copolymer modified nanochannel that may address the challenge. In the tricomponent poly(N-isopropylacrylamide) (PNIPAAm, PNI)-based copolymer system, phenylthiourea was used to bind the phosphate units of nucleotides and phenylboronic acid was introduced to combine the pentose ring of the nucleoside unit. Besides, a -COOH group with electron-withdrawing property was added into the phenylthiourea units, which may promote the hydrogen-bond-donating ability of thiourea. Specially, the smart copolymer not only provided static binding sites for recognition but also translated the recognition of ATP into their dynamic conformational transitions by changing the hydrogen-bonding environments surrounding PNIPAAm chains, thus achieving the gating function of nanochannel, which resembled the integration and coordination of Kir6.x and SUR units in active KATP. The ATP-regulated ion channel exhibited excellent stability and reversibility. This study is the first example showing how to learn from nature to assemble the ATP-responsive artificial nanochannel and demonstrate the possible mechanism of ATP gating.


Assuntos
Trifosfato de Adenosina , Biomimética , Sítios de Ligação , Nucleotídeos , Receptores de Sulfonilureias
5.
Front Cell Infect Microbiol ; 11: 771010, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34804999

RESUMO

Synonymous codon usage bias is a universal characteristic of genomes across various organisms. Autophagy-related gene 13 (atg13) is one essential gene for autophagy initiation, yet the evolutionary trends of the atg13 gene at the usages of nucleotide and synonymous codon remains unexplored. According to phylogenetic analyses for the atg13 gene of 226 eukaryotic organisms at the nucleotide and amino acid levels, it is clear that their nucleotide usages exhibit more genetic information than their amino acid usages. Specifically, the overall nucleotide usage bias quantified by information entropy reflected that the usage biases at the first and second codon positions were stronger than those at the third position of the atg13 genes. Furthermore, the bias level of nucleotide 'G' usage is highest, while that of nucleotide 'C' usage is lowest in the atg13 genes. On top of that, genetic features represented by synonymous codon usage exhibits a species-specific pattern on the evolution of the atg13 genes to some extent. Interestingly, the codon usages of atg13 genes in the ancestor animals (Latimeria chalumnae, Petromyzon marinus, and Rhinatrema bivittatum) are strongly influenced by mutation pressure from nucleotide composition constraint. However, the distributions of nucleotide composition at different codon positions in the atg13 gene display that natural selection still dominates atg13 codon usages during organisms' evolution.


Assuntos
Uso do Códon , Nucleotídeos , Animais , Autofagia/genética , Eucariotos/genética , Evolução Molecular , Nucleotídeos/genética , Filogenia
6.
Int J Mol Sci ; 22(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34639127

RESUMO

Toxoplasma gondii is an apicomplexan parasite that infects and proliferates within many different types of host cells and infects virtually all warm-blooded animals and humans. Trypanosoma brucei is an extracellular kinetoplastid that causes human African trypanosomiasis and Nagana disease in cattle, primarily in rural sub-Saharan Africa. Current treatments against both parasites have limitations, e.g., suboptimal efficacy and adverse side effects. Here, we investigate the potential cellular and molecular targets of a trithiolato-bridged arene ruthenium complex conjugated to 9-(2-hydroxyethyl)-adenine (1), which inhibits both parasites with IC50s below 10-7 M. Proteins that bind to 1 were identified using differential affinity chromatography (DAC) followed by shotgun-mass spectrometry. A trithiolato-bridged ruthenium complex decorated with hypoxanthine (2) and 2-hydroxyethyl-adenine (3) were included as controls. Transmission electron microscopy (TEM) revealed distinct ultrastructural modifications in the mitochondrion induced by (1) but not by (2) and (3) in both species. DAC revealed 128 proteins in T. gondii and 46 proteins in T. brucei specifically binding to 1 but not 2 or 3. In T. gondii, the most abundant was a protein with unknown function annotated as YOU2. This protein is a homolog to the human mitochondrial inner membrane translocase subunit Tim10. In T. brucei, the most abundant proteins binding specifically to 1 were mitochondrial ATP-synthase subunits. Exposure of T. brucei bloodstream forms to 1 resulted in rapid breakdown of the ATP-synthase complex. Moreover, both datasets contained proteins involved in key steps of metabolism and nucleic acid binding proteins.


Assuntos
Nucleotídeos/química , Compostos de Rutênio/farmacologia , Compostos de Sulfidrila/química , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase/tratamento farmacológico , Humanos , Membranas Mitocondriais/efeitos dos fármacos , Membranas Mitocondriais/metabolismo , Proteínas de Protozoários/metabolismo , Compostos de Rutênio/química , Toxoplasma/metabolismo , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase/metabolismo , Tripanossomíase/parasitologia
7.
BMC Genomics ; 22(1): 755, 2021 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-34674653

RESUMO

BACKGROUND: Mitochondrial genomes (mitogenomes) have greatly improved our understanding of the backbone phylogeny of Lepidoptera, but few studies on comparative mitogenomics below the family level have been conducted. Here, we generated 13 mitogenomes of eight tortricid species, reannotated 27 previously reported mitogenomes, and systematically performed a comparative analysis of nucleotide composition, gene variation and phylogenetic performance. RESULTS: The lengths of completely sequenced mitogenomes ranged from 15,440 bp to 15,778 bp, and the gene content and organization were conserved in Tortricidae and typical for Lepidoptera. Analyses of AT-skew and GC-skew, the effective number of codons and the codon bias index all show a base bias in Tortricidae, with little heterogeneity among the major tortricid groups. Variations in the divergence rates among 13 protein-coding genes of the same tortricid subgroup and of the same PCG among tortricid subgroups were detected. The secondary structures of 22 transfer RNA genes and two ribosomal RNA genes were predicted and comparatively illustrated, showing evolutionary heterogeneity among different RNAs or different regions of the same RNA. The phylogenetic uncertainty of Enarmoniini in Tortricidae was confirmed. The synonymy of Bactrini and Olethreutini was confirmed for the first time, with the representative Bactrini consistently nesting in the Olethreutini clade. Nad6 exhibits the highest phylogenetic informativeness from the root to the tip of the resulting tree, and the combination of the third coding positions of 13 protein-coding genes shows extremely high phylogenetic informativeness. CONCLUSIONS: This study presents 13 mitogenomes of eight tortricid species and represents the first detailed comparative mitogenomics study of Tortricidae. The results further our understanding of the evolutionary architectures of tortricid mitogenomes and provide a basis for future studies of population genetics and phylogenetic investigations in this group.


Assuntos
Genoma Mitocondrial , Mariposas , Animais , Mariposas/genética , Nucleotídeos/genética , Filogenia , RNA Ribossômico/genética , RNA de Transferência/genética
8.
Enzymes ; 49: 215-233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696833

RESUMO

RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of processive nucleic acid polymerases, carrying out DNA-independent replication/transcription processes. Although viral RdRPs have versatile global structures, they do share a structurally highly conserved active site comprising catalytic motifs A-G. In spite of different initiation modes, the nucleotide addition cycle (NAC) in the RdRP elongation phase probably follows consistent mechanisms. In this chapter, representative structures of picornavirus RdRP elongation complexes are used to illustrate RdRP NAC mechanisms. In the pre-chemistry part of the NAC, RdRPs utilize a unique palm domain-based active site closure that can be further decomposed into two sequential steps. In the post-chemistry part of the NAC, the translocation process is stringently controlled by the RdRP-specific motif G, resulting in asymmetric movements of the template-product RNA. Future efforts to elucidate regulation/intervention mechanisms by mismatched NTPs or nucleotide analog antivirals are necessary to achieve comprehensive understandings of viral RdRP NAC.


Assuntos
Picornaviridae , RNA Polimerase Dependente de RNA , Domínio Catalítico , Nucleotídeos , Picornaviridae/genética , RNA , RNA Polimerase Dependente de RNA/genética
9.
Enzymes ; 49: 39-62, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696838

RESUMO

Nucleotide analogs are the cornerstone of direct acting antivirals used to control infection by RNA viruses. Here we review what is known about existing nucleotide/nucleoside analogs and the kinetics and mechanisms of RNA and DNA replication, with emphasis on the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in comparison to HIV reverse transcriptase and Hepatitis C RdRp. We demonstrate how accurate kinetic analysis reveals surprising results to explain the effectiveness of antiviral nucleoside analogs providing guidelines for the design of new inhibitors.


Assuntos
COVID-19 , Hepatite C Crônica , Monofosfato de Adenosina , Alanina , Antivirais/farmacologia , Humanos , Cinética , Nucleotídeos , RNA Viral/genética , SARS-CoV-2
10.
Anal Chim Acta ; 1182: 338943, 2021 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-34602188

RESUMO

PIK3CA H1047R gene plays an important role in the PI3K/Akt/mTOR signaling pathway, and its mutation is closely related to the occurrence and development of breast cancer and Lipoblastoma. Therefore, it is of great value to detect the PIK3CA H1047R mutant gene. Here, an analytical method coupled CRISPR/Cas12a with rolling circle amplification (RCA) technology was constructed for ultra-sensitive and specific detection of the single-nucleotide variant (SNV) of the PIK3CA H1047R gene. With efficient amplification of RCA and CRISPR/Cas12a, the detection limit of the mutant target and mixture of the mutant with wild-type target were as low as 10 aM and 0.036%, respectively. The detection limit of the RCA-CRISPR/Cas12a method was lower than that of allelic specific PCR (AS-PCR) for detecting SNV of the PIK3CA H1047R gene. Hence, this RCA-CRISPR/Cas12a method is sensitive and specific for the detection of SNV. What's more, this strategy provides a new idea for medical diagnosis and lays a technical foundation for the research of PI3K/Akt/mTOR signaling pathways.


Assuntos
Sistemas CRISPR-Cas , Fosfatidilinositol 3-Quinases , Sistemas CRISPR-Cas/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Humanos , Mutação , Nucleotídeos , Fosfatidilinositol 3-Quinases/genética
11.
Anal Chem ; 93(43): 14568-14576, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34672523

RESUMO

Single-nucleotide polymorphisms (SNPs) are important hallmarks of human diseases. Herein, we develop a single quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of SNPs in cancer cells. This assay involves only a linear padlock probe for MPG-RCA. The presence of a mutant target facilitates the circularization of linear padlock probes to initiate RCA, producing three short single-stranded DNAs (ssDNAs) with the assistance of nicking endonuclease. The resulting ssDNAs can function as primers to induce cyclic MPG-RCA, resulting in the exponential amplification and generation of large numbers of linker probes. The linker probes can subsequently hybridize with the Cy5-labeled reporter probes and the biotinylated capture probes to obtain the sandwich hybrids. The assembly of these sandwich hybrids on the 605 nm-emission quantum dot (605QD) generates the 605QD-oligonucleotide-Cy5 nanostructures, resulting in efficient FRET from the 605QD to Cy5. This nanosensor is free from both the complicated probe design and the exogenous primers and has distinct advantages of high amplification efficiency, zero background signal, good specificity, and high sensitivity. It can detect SNPs with a large dynamic range of 8 orders of magnitude and a detection limit of 5.41 × 10-20 M. Moreover, this nanosensor can accurately distinguish as low as 0.001% mutation level from the mixtures, which cannot be achieved by previously reported methods. Furthermore, it can discriminate cancer cells from normal cells and even quantify SNP at the single-cell level.


Assuntos
Neoplasias , Pontos Quânticos , DNA de Cadeia Simples , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico , Nucleotídeos , Polimorfismo de Nucleotídeo Único
12.
Enzymes ; 49: 315-354, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34696837

RESUMO

The treatment of viral infections remains challenging, in particular in the face of emerging pathogens. Broad-spectrum antiviral drugs could potentially be used as a first line of defense. The RNA-dependent RNA polymerase (RdRp) of RNA viruses serves as a logical target for drug discovery and development efforts. Herein we discuss compounds that target RdRp of poliovirus, hepatitis C virus, influenza viruses, respiratory syncytial virus, and the growing data on coronaviruses. We focus on nucleotide analogs and mechanisms of action and resistance.


Assuntos
Antivirais/farmacologia , Nucleotídeos/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Proteínas do Complexo da Replicase Viral/antagonistas & inibidores , Vírus/enzimologia , Replicação Viral
13.
Cell Mol Life Sci ; 78(21-22): 6979-6993, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34596691

RESUMO

Among alternative splicing events in the human transcriptome, tandem NAGNAG acceptor splice sites represent an appreciable proportion. Both proximal and distal NAG can be used to produce two splicing isoforms differing by three nucleotides. In some cases, the upstream exon can be alternatively spliced as well, which further increases the number of possible transcripts. In this study, we showed that NAG choice in tandem splice site depends considerably not only on the concerned acceptor, but also on the upstream donor splice site sequence. Using an extensive set of experiments with systematically modified two-exonic minigene systems of AFAP1L2 or CSTD gene, we recognized the third and fifth intronic upstream donor splice site position and the tandem acceptor splice site region spanning from -10 to +2, including NAGNAG itself, as the main drivers. In addition, competition between different branch points and their composition were also shown to play a significant role in NAG choice. All these nucleotide effects appeared almost additive, which explained the high variability in proximal versus distal NAG usage.


Assuntos
Processamento Alternativo/genética , Nucleotídeos/genética , Sítios de Splice de RNA/genética , Sequências de Repetição em Tandem/genética , Linhagem Celular Tumoral , Éxons/genética , Células HeLa , Humanos , Íntrons/genética
14.
Anal Chem ; 93(45): 14955-14965, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34694783

RESUMO

In the context of the recent pandemic, the necessity of inexpensive and easily accessible rapid-test kits is well understood and need not be stressed further. In light of this, we report a multi-nucleotide probe-based diagnosis of SARS-CoV-2 using a bioelectronics platform, comprising low-cost chemiresistive biochips, a portable electronic readout, and an Android application for data acquisition with machine-learning-based decision making. The platform performs the desired diagnosis from standard nasopharyngeal and/or oral swabs (both on extracted and non-extracted RNA samples) without amplifying the viral load. Being a reverse transcription polymerase chain reaction-free hybridization assay, the proposed approach offers inexpensive, fast (time-to-result: ≤ 30 min), and early diagnosis, as opposed to most of the existing SARS-CoV-2 diagnosis protocols recommended by the WHO. For the extracted RNA samples, the assay accounts for 87 and 95.2% test accuracies, using a heuristic approach and a machine-learning-based classification method, respectively. In case of the non-extracted RNA samples, 95.6% decision accuracy is achieved using the heuristic approach, with the machine-learning-based best-fit model producing 100% accuracy. Furthermore, the availability of the handheld readout and the Android application-based simple user interface facilitates easy accessibility and portable applications. Besides, by eliminating viral RNA extraction from samples as a pre-requisite for specific detection, the proposed approach presents itself as an ideal candidate for point-of-care SARS-CoV-2 diagnosis.


Assuntos
COVID-19 , SARS-CoV-2 , Inteligência Artificial , Teste para COVID-19 , Humanos , Nucleotídeos , RNA Viral/genética , Sensibilidade e Especificidade
15.
Elife ; 102021 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-34617885

RESUMO

The absence of 'shovel-ready' anti-coronavirus drugs during vaccine development has exceedingly worsened the SARS-CoV-2 pandemic. Furthermore, new vaccine-resistant variants and coronavirus outbreaks may occur in the near future, and we must be ready to face this possibility. However, efficient antiviral drugs are still lacking to this day, due to our poor understanding of the mode of incorporation and mechanism of action of nucleotides analogs that target the coronavirus polymerase to impair its essential activity. Here, we characterize the impact of remdesivir (RDV, the only FDA-approved anti-coronavirus drug) and other nucleotide analogs (NAs) on RNA synthesis by the coronavirus polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. We reveal that the location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We show that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into backtrack as far as 30 nt, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this NA well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , RNA-Polimerase RNA-Dependente de Coronavírus/antagonistas & inibidores , Nucleotídeos/farmacologia , SARS-CoV-2/efeitos dos fármacos , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Linhagem Celular , RNA-Polimerase RNA-Dependente de Coronavírus/metabolismo , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Humanos , Modelos Teóricos , Nucleotídeos/metabolismo , RNA Viral , SARS-CoV-2/enzimologia , Processos Estocásticos , Replicação Viral/efeitos dos fármacos
16.
Artigo em Inglês | MEDLINE | ID: mdl-34682349

RESUMO

Drug-induced liver injury (DILI) is a major cause of drug development failure and drug withdrawal from the market after approval. The identification of human risk factors associated with susceptibility to DILI is of paramount importance. Increasing evidence suggests that genetic variants may lead to inter-individual differences in drug response; however, individual single-nucleotide polymorphisms (SNPs) usually have limited power to predict human phenotypes such as DILI. In this study, we aim to identify appropriate statistical methods to investigate gene-gene and/or gene-environment interactions that impact DILI susceptibility. Three machine learning approaches, including Multivariate Adaptive Regression Splines (MARS), Multifactor Dimensionality Reduction (MDR), and logistic regression, were used. The simulation study suggested that all three methods were robust and could identify the known SNP-SNP interaction when up to 4% of genotypes were randomly permutated. When applied to a real-life DILI chronicity dataset, both MARS and MDR, but not logistic regression, identified combined genetic variants having better associations with DILI chronicity in comparison to the use of individual SNPs. Furthermore, a simple decision tree model using the SNPs identified by MARS and MDR was developed to predict DILI chronicity, with fair performance. Our study suggests that machine learning approaches may help identify gene-gene interactions as potential risk factors for better assessing complicated diseases such as DILI chronicity.


Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Doença Hepática Induzida por Substâncias e Drogas/genética , Humanos , Aprendizado de Máquina , Nucleotídeos , Polimorfismo de Nucleotídeo Único , Fatores de Risco
17.
J Phys Chem B ; 125(42): 11660-11672, 2021 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-34652157

RESUMO

Targeting mismatched base pairs containing DNA using small molecules and exploring the underlying mechanism involved during the binding interactions is one of the fundamental aspects of drug design. These molecules in turn are used in nucleic acid targeted therapeutics and cancer diagnosis. In this work, we systematically delineate the binding of the anticancer drug, epirubicin hydrochloride (EPR) with 20-mer duplex DNA, having both natural nucleobase pairing and thermodynamically least stable non-Watson-Crick base pairing. From the thermal denaturation studies, we observed that EPR can remarkably enhance the thermal stability of cytosine-cytosine (CC) and cytosine-thymine (CT) mismatched (MM) DNA over other 20-mer duplex DNA. From steady-state fluorescence spectroscopy and isothermal titration calorimetry studies, we concluded that EPR binds strongly with the mismatched duplex DNA through the intercalation binding mode. The interaction of EPR and duplex DNA has also been monitored at a single molecular resolution using fluorescence correlation spectroscopy (FCS). Dynamic quantitates such as diffusion coefficients and hydrodynamic radii obtained from an FCS study along with association and dissociation rate constants estimated from intensity time trace analyses further substantiate the stronger binding affinity of EPR to the thermally less stable mismatched DNA, formed by the most discriminating nucleobase (viz. cytosine). Additionally, we have shown that EPR can be sequestered from nucleic acids using a mixed micellar system of an anionic surfactant and a triblock copolymer. From thermal denaturation studies and circular dichroism spectroscopy, we found that the extent of drug sequestration depends on the binding affinity of EPR to the duplex DNA, and this mixed micellar system can be employed for the removal of excess drug in the case of a drug overdose.


Assuntos
Micelas , Nucleotídeos , Pareamento de Bases , DNA , Epirubicina , Conformação de Ácido Nucleico , Termodinâmica
18.
Molecules ; 26(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34684811

RESUMO

Traditional glycosyltransferase (GT) activity assays are not easily configured for rapid detection nor for high throughput screening because they rely on radioactive product isolation, the use of heterogeneous immunoassays or mass spectrometry. In a typical glycosyltransferase biochemical reaction, two products are generated, a glycosylated product and a nucleotide released from the sugar donor substrate. Therefore, an assay that detects the nucleotide could be universal to monitor the activity of diverse glycosyltransferases in vitro. Here we describe three homogeneous and bioluminescent glycosyltransferase activity assays based on UDP, GDP, CMP, and UMP detection. Each of these assays are performed in a one-step detection that relies on converting the nucleotide product to ATP, then to bioluminescence using firefly luciferase. These assays are highly sensitive, robust and resistant to chemical interference. Various applications of these assays are presented, including studies on the specificity of sugar transfer by diverse GTs and the characterization of acceptor substrate-dependent and independent nucleotide-sugar hydrolysis. Furthermore, their utility in screening for specific GT inhibitors and the study of their mode of action are described. We believe that the broad utility of these nucleotide assays will enable the investigation of a large number of GTs and may have a significant impact on diverse areas of Glycobiology research.


Assuntos
Glicosiltransferases/antagonistas & inibidores , Glicosiltransferases/metabolismo , Medições Luminescentes/métodos , Trifosfato de Adenosina/metabolismo , Animais , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicômica/métodos , Ensaios de Triagem em Larga Escala/métodos , Cinética , Luciferases de Vaga-Lume/metabolismo , Nucleotídeos/metabolismo , Especificidade por Substrato
19.
ACS Nano ; 15(10): 16642-16653, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34618430

RESUMO

We describe a method for direct tRNA sequencing using the Oxford Nanopore MinION. The principal technical advance is custom adapters that facilitate end-to-end sequencing of individual transfer RNA (tRNA) molecules at subnanometer precision. A second advance is a nanopore sequencing pipeline optimized for tRNA. We tested this method using purified E. coli tRNAfMet, tRNALys, and tRNAPhe samples. 76-92% of individual aligned tRNA sequence reads were full length. As a proof of concept, we showed that nanopore sequencing detected all 43 expected isoacceptors in total E. coli MRE600 tRNA as well as isodecoders that further define that tRNA population. Alignment-based comparisons between the three purified tRNAs and their synthetic controls revealed systematic nucleotide miscalls that were diagnostic of known modifications. Systematic miscalls were also observed proximal to known modifications in total E. coli tRNA alignments, including a highly conserved pseudouridine in the T loop. This work highlights the potential of nanopore direct tRNA sequencing as well as improvements needed to implement tRNA sequencing for human healthcare applications.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Nucleotídeos
20.
Arthritis Res Ther ; 23(1): 251, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593020

RESUMO

BACKGROUND: Hypertrophy of ligamentum flavum (HLF) is a common lumbar degeneration disease (LDD) with typical symptoms of low back pain and limb numbness owing to an abnormal pressure on spinal nerves. Previous studies revealed HLF might be caused by fibrosis, inflammatory, and other bio-pathways. However, a global analysis of HLF is needed severely. METHODS: A genome-wide DNA methylation and single-nucleotide polymorphism analysis were performed from five LDD patients with HLF and five LDD patients without HLF. Comprehensive integrated analysis was performed using bioinformatics analysis and the validated experiments including Sanger sequencing, methylation-specific PCR, qPCR and ROC analysis. Furthermore, the function of novel genes in ligamentum flavum cells (LFCs) was detected to explore the molecular mechanism in HLF through knock down experiment, overexpression experiment, CCK8 assay, apoptosis assay, and so on. RESULTS: We identified 69 SNP genes and 735 661 differentially methylated sites that were enriched in extracellular matrix, inflammatory, and cell proliferation. A comprehensive analysis demonstrated key genes in regulating the development of HLF including ACSM5. Furthermore, the hypermethylation of ACSM5 that was mediated by DNMT1 led to downregulation of ACSM5 expression, promoted the proliferation and fibrosis, and inhibited the apoptosis of LFCs. CONCLUSION: This study revealed that DNMT1/ACSM5 signaling could enhance HLF properties in vitro as a potential therapeutic strategy for HLF.


Assuntos
Ligamento Amarelo , Estenose Espinal , Coenzima A Ligases , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/genética , Humanos , Hipertrofia/genética , Ligamento Amarelo/metabolismo , Vértebras Lombares , Nucleotídeos/metabolismo , Estenose Espinal/genética , Estenose Espinal/metabolismo
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