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1.
Nanoscale ; 13(29): 12687-12696, 2021 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-34477619

RESUMO

Measuring the electrophoretic mobility of molecules is a powerful experimental approach for investigating biomolecular processes. A frequent challenge in the context of single-particle measurements is throughput, limiting the obtainable statistics. Here, we present a molecular force sensor and charge detector based on parallelised imaging and tracking of tethered double-stranded DNA functionalised with charged nanoparticles interacting with an externally applied electric field. Tracking the position of the tethered particle with simultaneous nanometre precision and microsecond temporal resolution allows us to detect and quantify the electrophoretic force down to the sub-piconewton scale. Furthermore, we demonstrate that this approach is suitable for detecting changes to the particle charge state, as induced by the addition of charged biomolecules or changes to pH. Our approach provides an alternative route to studying structural and charge dynamics at the single molecule level.


Assuntos
Nanopartículas , Nanotecnologia , DNA , Eletroforese
2.
Nanoscale ; 13(32): 13746-13757, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34477649

RESUMO

Manipulation of temperature can be used to actuate DNA origami nano-hinges containing gold nanoparticles. We develop a physical model of this system that uses partition function analysis of the interaction between the nano-hinge and nanoparticle to predict the probability that the nano-hinge is open at a given temperature. The model agrees well with experimental data and predicts experimental conditions that allow the actuation temperature of the nano-hinge to be tuned over a range of temperatures from 30 °C to 45 °C. Additionally, the model identifies microscopic interactions that are important to the macroscopic behavior of the system, revealing surprising features of the system. This combination of physical insight and predictive potential is likely to inform future designs that integrate nanoparticles into dynamic DNA origami structures or use strand binding interactions to control dynamic DNA origami behavior. Furthermore, our modeling approach could be expanded to consider the incorporation, stability, and actuation of other types of functional elements or actuation mechanisms integrated into nucleic acid devices.


Assuntos
Ouro , Nanopartículas Metálicas , DNA , Conformação de Ácido Nucleico , Temperatura
3.
Nanoscale ; 13(32): 13758-13763, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34477650

RESUMO

Here, we report how the nature of the hydrophobic core affects the molecular interactions of DNA block copolymer assemblies. Three different amphiphilic DNA block copolymers, DNA-b-polystyrene (DNA-b-PS), DNA-b-poly(2-vinylpyridine) (DNA-b-P2VP), and DNA-b-poly(methyl acrylate) (DNA-b-PMA) were synthesized and assembled into spherical micelles composed of a hydrophobic polymer core and DNA corona. Interestingly, DNA block copolymer micelles having different hydrophobic cores exhibited markedly different molecular and biological interactions. DNA-b-PS exhibited higher melting temperature, sharper melting transition, higher stability to nuclease-catalyzed DNA degradation, and higher cellular uptake efficiency compared to DNA-b-P2VP and DNA-b-PMA. The investigation of the self-assembly behavior revealed a much higher aggregation number and DNA density for DNA-b-PS micelles, which explains the superior properties of DNA-b-PS. These results demonstrate that the type of the hydrophobic core polymer, which has been largely overlooked, has a profound impact on the molecular and biological interactions of the DNA shell.


Assuntos
Micelas , Polímeros , DNA , Interações Hidrofóbicas e Hidrofílicas , Poliestirenos
4.
Nanoscale ; 13(32): 13795-13808, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34477654

RESUMO

Light-activated functional materials capable of remote control over duplex and G-quadruplex (G4) nucleic acids formation at the cellular level are still very rare. Herein, we report on the photoinduced macrocyclisation of a helicenoid quinoline derivative of binaphthol that selectively provides easy access to an unprecedented class of extended heteroaromatic structures with remarkable photophysical and DNA/RNA binding properties. Thus, while the native bisquinoline precursor shows no DNA binding activity, the new in situ photochemically generated probe features high association constants to DNA and RNA G4s. The latter inhibits DNA synthesis by selectively stabilizing G4 structures associated with oncogenic promoters and telomere repeat units. Finally, the light sensitive compound is capable of in cellulo photoconversion, localizes primarily in the G4-rich sites of cancer cells, competes with a well-known G4 binder and shows a clear nuclear co-localization with the quadruplex specific antibody BG4. This work provides a benchmark for the future design and development of a brand-new generation of light-activated target-selective G4-binders.


Assuntos
Corantes Fluorescentes , Quadruplex G , DNA , Ligantes , Telômero
5.
Nanoscale ; 13(33): 14147-14155, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34477696

RESUMO

The potential of carbon dots (CDs) for photonic conversion to charged states, together with the ability of DNA to transport such charge for extensive charge separation, offers an opportunity to control directionality of migration for photo-induced radical cations in CD-DNA based nano-assemblies. This is achieved through engineering the reaction valency of CDs whereby one CD is covalently conjugated with one ssDNA strand. Subsequently, a CD-DNA-CD nano-dumbbell architecture was created through hybridization mediated self-assembly. The time and intensity-dependent transduction of visible light photonic energy to chemical potential in DNA was achieved through irradiation of 1,4-diaminoathraquinone and glyoxal derived CD with 100 W tungsten source and natural sunlight. Following charge injection by CD, the radical cation migration in DNA was perceived through trapping of the hole in repeated GG steps in the DNA. Overall, a breakthrough in visible-light-induced charge transfer by CD into DNA was achieved, potentially applicable to optobioelectronics.


Assuntos
Carbono , Pontos Quânticos , DNA , Luz , Hibridização de Ácido Nucleico
6.
Nanoscale ; 13(30): 12848-12853, 2021 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-34477769

RESUMO

Nucleic acid nanostructures are promising biomaterials for the delivery of homologous gene therapy drugs. Herein, we report a facile strategy for the construction of target mRNA (scaffold) and antisense (staple strands) co-assembled RNA/DNA hybrid "origami" for efficient gene therapy. In our design, the mRNA was folded into a chemically well-defined nanostructure through RNA-DNA hybridization with high yield. After the incorporation of an active cell-targeting aptamer, the tailored RNA/DNA hybrid origami demonstrated efficient cellular uptake and controllable release of antisenses in response to intracellular RNase H digestion. The biocompatible RNA/DNA origami (RDO) elicited a noticeable inhibition of cell proliferation based on the silencing of the tumor-associated gene polo-like kinase 1 (PLK1). This RDO-based nanoplatform provides a novel strategy for the further development of gene therapy.


Assuntos
Nanoestruturas , RNA , DNA/genética , Terapia Genética , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , RNA/genética
7.
Anal Chim Acta ; 1178: 338811, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482872

RESUMO

Capillary gel electrophoresis is widely applied for determination of sequence and size of DNA, in which the sieving gel plays an unignorable role. Herein, a pore-size controllable hydrogel was synthesized in the capillary with two symmetrical tetrahedron-like macromonomers consisting of pentaerythritoltetra (succinimidylcarboxypentyl) polyoxyethylene (PS) and pentaerythritoltetra (aminopropyl) polyoxyethylene) (PA). By capillary electrophoresis of the DNA fragments with this hydrogel, it is found that a homogenous structure of hydrogel which is more suitable for the DNA separation can be achieved when the molecular weight of PA is approximate to that of PS. DNA fragments smaller than 1500 bp can be well resolved in this hydrogel within 13 min. More than 100 consecutive runs can be carried out in such a dynamically coated capillary before performance begins to degrade. Notably, such hydrogel can realize separation of dsDNA up to single base pair resolution and same length of dsDNA with 1 bp difference.


Assuntos
Hidrogéis , Polietilenoglicóis , DNA , Eletroforese Capilar , Peso Molecular
8.
Anal Chim Acta ; 1178: 338762, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34482873

RESUMO

We report the synthesis and characterization of a new hybrid magnetic composite formed by the enveloping of magnetic iron oxide nanoparticles (γ-NP) with chains of the conductive polymer PEDOT, and its use for the efficient separation of DNA molecules from complex biological samples, allowing the high yield separation of a pure and high-quality DNA fraction. The successful formation of the γ-NP/PEDOT composite was confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, UV visible spectroscopy (UV-Vis), and magnetic hysteresis loop measurements. The nanocomposites showed an excellent capacity of DNA adsorption (Qe âˆ¼ 248 mg/g) in a model system consisting of salmon sperm DNA. When the γ-NP/PEDOT was used in protocols to extract the DNA from complex samples, the corresponding yield was in the range of 6.4 µg (blood) and 7.3 µg (bacteria), as evaluated quality by UV-Vis, PCR analysis, and electrophoresis assays. We also established that the captured DNA does not need to be detached from the nanocomposite for use as seeding material in PCR amplification experiments. These results and the simplicity of the protocols indicate that the γ-NP/PEDOT composite is a promising DNA absorbent, being competitive with the commercially available magnetic purification kits.


Assuntos
Nanocompostos , Compostos Bicíclicos Heterocíclicos com Pontes , DNA/genética , Polímeros , Espectroscopia de Infravermelho com Transformada de Fourier
9.
Anal Chem ; 93(36): 12346-12352, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34469684

RESUMO

Synthetic DNA walkers are artificially designed DNA self-assemblies with the capability of performing quasi-mechanical movement at the micro/nanoscale and have shown extensive promise in biosensing, intracellular imaging, and drug delivery. However, DNA walkers are usually constructed by covalently or coordinately binding DNA strands specifically to hard surfaces, thereby greatly limiting their movement efficiency. Herein, we report an intraparticle and interparticle transferable DNA walker (dynamic micelle-supported DNA walker, DM-walker) constructed by immobilizing walking tracks and walking arms onto the corona of DNA micelles according to the principle of Watson-Crick base pairing. The DNAzyme-powered walking arm can drive the intraparticle and interparticle movements of the DM-walker due to the fact that the dynamic structure of the DNA micelle helps overcome the spatial barrier between the arms and tracks in the system, resulting in high walking efficiency. Moreover, the whole DM-walker can be constructed by self-assembly, getting rid of the tedious process and low efficiency of fixing DNA strands on hard surfaces. Taking miRNA-10b as a model target, the DM-walker demonstrates high walking efficiency (reaction duration of 20 min) and high sensitivity (LOD of 87 pM). The proposed DM-walker provides an avenue to develop novel DNA walkers on dynamic interfaces and holds great potential in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , MicroRNAs , DNA , Limite de Detecção , Micelas , Andadores
10.
Anal Chem ; 93(36): 12329-12336, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34474564

RESUMO

"On-demand" accurate imaging of multiple intracellular miRNAs will significantly improve the detection reliability and accuracy. However, the "always-active" design of traditional multicomponent detection probes enables them to passively recognize and output signals as soon as they encounter targets, which will inevitably impair the detection accuracy and, inevitably, result in false-positive signals. To address this scientific problem, in this work, we developed a near-infrared (NIR) light-activated multicomponent detection intelligent nanoprobe for spatially and temporally controlled on-demand accurate imaging of multiple intracellular miRNAs. The proposed intelligent nanoprobe is composed of a rationally designed UV light-responsive triangular DNA nano sucker (TDS) and upconversion nanoparticles (UCNPs), named UCNPs@TDS (UTDS), which can enter cells autonomously through endocytosis and enable remote regulation of on-demand accurate imaging for multiple intracellular miRNAs using NIR light illumination at a chosen time and place. It is worth noting that the most important highlight of the UTDS we designed in this work is that it can resist nonspecific activation as well as effectively avoid false-positive signals and improve the accuracy of imaging of multiple intracellular miRNAs. Moreover, distinguishing different kinds of cell lines with different miRNA expressions levels can be also achieved through this NIR light-activated intelligent UTDS, showing feasible prospects in precise imaging and disease diagnosis.


Assuntos
MicroRNAs , Nanopartículas , DNA , Raios Infravermelhos , Reprodutibilidade dos Testes
11.
Nat Commun ; 12(1): 5201, 2021 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-34465779

RESUMO

N6-methyladenosine (m6A) is a post-transcriptional modification that controls gene expression by recruiting proteins to RNA sites. The modification also slows biochemical processes through mechanisms that are not understood. Using temperature-dependent (20°C-65°C) NMR relaxation dispersion, we show that m6A pairs with uridine with the methylamino group in the anti conformation to form a Watson-Crick base pair that transiently exchanges on the millisecond timescale with a singly hydrogen-bonded low-populated (1%) mismatch-like conformation in which the methylamino group is syn. This ability to rapidly interchange between Watson-Crick or mismatch-like forms, combined with different syn:anti isomer preferences when paired (~1:100) versus unpaired (~10:1), explains how m6A robustly slows duplex annealing without affecting melting at elevated temperatures via two pathways in which isomerization occurs before or after duplex annealing. Our model quantitatively predicts how m6A reshapes the kinetic landscape of nucleic acid hybridization and conformational transitions, and provides an explanation for why the modification robustly slows diverse cellular processes.


Assuntos
Adenosina/análogos & derivados , DNA/química , DNA/metabolismo , Adenosina/química , Adenosina/genética , Adenosina/metabolismo , Pareamento de Bases , DNA/genética , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Processamento Pós-Transcricional do RNA , Uridina/química , Uridina/genética , Uridina/metabolismo
12.
Talanta ; 235: 122694, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517578

RESUMO

This work reports a simple strategy for Candida auris genomic DNA (gDNA) detection, a multi-resistant fungus associated with nosocomial outbreaks in healthcare settings, presenting high mortality and morbidity rates. The platform was developed using gold electrode sensitized with specific DNA capture probe and ninhydrin as a novel DNA hybridization indicator. The genosensor was able to detect C. auris in urine sample by differential pulse voltammetry and electrochemical impedance spectroscopy. The biosensor's analytical performance was evaluated by differential pulse voltammetry, detecting up to 4.5 pg µL-1 of C. auris gDNA in urine (1:10, V/V). Moreover, the genosensor was reused eight times with no loss in the current signal response. The genosensor showed selectivity and stability, maintaining 100% of its response up to 80 days of storage. In order to analyze interactions of single and double-stranded DNA with ninhydrin, SEM, AFM and molecular dynamics studies followed by docking simulations were performed. Theoretical calculations showed ninhydrin interactions more favorably with dsDNA in an A-T rich binding pocket rather than with the ssDNA. Therefore, the proposed system is a promising electrochemical detection device towards a more accurate detection of C. auris gDNA in biological samples.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Candida/genética , DNA , Ninidrina
13.
Talanta ; 235: 122747, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517615

RESUMO

Microchip capillary electrophoresis (MCE) is a powerful technique for rapid separation; however, its acceptance in routine laboratories is still limited. Compromises caused by the efforts for solving different problems, such as reducing its cost of fabrication and ensuring high separation efficiency, undermine the competitiveness of this technology compared to other separation techniques. Contrary to the conventional pursuit of narrow microchannels, this study investigated the suitability of microchips with channels at the sub-millimeter level, targeting the simplification of the overall operation, cost reduction, and robustness improvement. To this effect, we considered the influence of pressurized flow and Joule heating on the separation. The suppression of pressurized flow with viscous solutions was confirmed through a combination of simulations and experimental results, indicating that the buffer viscosity was enough for successful separation. We fabricated channels of 200 µm × 230 µm using computer numerical controlled (CNC) machining and obtained theoretical plate numbers of 4.8 × 105 m-1 and 5.3 × 105 m-1 for fluorescein isothiocyanate (FITC) labeled small molecules and DNA fragments, respectively, with a buffer viscosity of 168 mPa s (0.5 % hydroxypropyl methylcellulose, HPMC). These values are comparable with that of narrow-bore microchips. Furthermore, we did not observe any deleterious effects with low-conductivity buffers. We investigated the rapid and highly sensitive detection of mycoplasma contamination and the real samples of circulating cell-free DNA (cfDNA), which gave a limit of detection (LOD) as low as 2.3 ng mL-1. Owing to the significant reduction in cost, ease of operation, and fast separation capabilities demonstrated in this work, MCE can be a viable alternative to the usual slab gel electrophoresis running in most biological laboratories.


Assuntos
Eletroforese em Microchip , DNA , Eletroforese Capilar , Derivados da Hipromelose , Limite de Detecção
14.
Talanta ; 235: 122749, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517617

RESUMO

Signal output mode is the important part of biosensor. In general, "signal on" and "signal off" are two common output modes. The development of dual signals-based ratio analysis as a powerful diagnostic tool has attracted widespread attention in the biosensor field in recent years. Dual signals ratio sensors with "signal on" and "signal off" are more favored because of their low background signal and better sensitivity and selectivity. In this study, inspired by the idea that EcoR V can cut specific sites of DNA to produce two corresponding fragments, and by using the capturing probe as guy wires, a reliable and sensitive method for EcoR V assay is developed based on the ratio of dual chemiluminescence (CL) signals for the first time. In particular, in the existence of the objective EcoR V, the substrate DNA would be degraded into two double stranded oligonucleotides with blunt ends which include the sequence I and the sequence II, then they can separately compete with two different corresponding capture probes on magnetic beads (MBs). One of capture probe hybridized with the sequence I containing more guanine (G) bases that reacted with the phenylglyoxal (PG) to produce chemical reaction which triggered a positive CL signal output I + CL as "signal-on"; another capture probe is priority to hybridize the sequence II, which triggered the weaker reporter DNA linked with horseradish peroxidase (HRP) probe to fall off the MBs, thereby outputting a negative CL signal I-CL as "signal-off". By comparing the linear relation and the correlation coefficient, the I-CL/I + CL ratio method has better linear relation (0.01-10 U/mL) and higher sensitivity (0.0045 U/mL). In addition, this developed strategy of high selectivity which can directly detect low concentration of target EcoR V in human serum, and thus this dual ratio biosensor might offer a promising detection approach for clinical diagnostics.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples , DNA/genética , DNA de Cadeia Simples/genética , Peroxidase do Rábano Silvestre , Humanos , Luminescência
15.
Talanta ; 235: 122805, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517663

RESUMO

DNA glycosylases can initiate base excision repair pathway to repair endogenous DNA base damages for the maintenance of genome stability. Multiple DNA glycosylases exhibit abnormal in various diseases, and the simultaneous measurement of different DNA glycosylases is critical to clinical diagnosis and drug discovery. Herein, we take advantage of single-molecule detection and bidirectional strand displacement amplification (SDA) to simultaneously detect uracil DNA glycolase (UDG) and human alkyladenine DNA glycosylase (hAAG). We design a partial double-stranded DNA (dsDNA) substrate modified with specific recognition sites of UDG and hAAG. The dsDNA substrate is labeled with BHQ1 and BHQ2 at the 5'-ends and then hybridizes with the Cy3/Cy5-labeled reporter probes to obtain the BHQ1/Cy3 and BHQ2/Cy5 base pairs, resulting in the quenching of Cy3/Cy5 fluorescence by BHQ1/BHQ2 via fluorescence resonance energy transfer (FRET). When UDG and hAAG are present, they can induce the base excision repair reaction and subsequently initiate the bidirectional SDA amplification process, releasing the Cy5/Cy3-labeled reporter probes from the dsDNA substrate and consequently the recovery of Cy5 and Cy3 fluorescence, which can be measured by single-molecule detection, with Cy5 indicating UDG and Cy3 indicating hAAG. This method possesses high sensitivity and good selectivity with the capability of quantifying multiple DNA glycosylases at the single-cell level. Furthermore, it can be used to simultaneously screen DNA glycosylase inhibitors and determine enzyme kinetic parameters, with the potential of sensing various DNA/RNA enzymes by simple changing the recognition sites of DNA substrates.


Assuntos
Reparo do DNA , Uracila-DNA Glicosidase , Bioensaio , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Uracila-DNA Glicosidase/metabolismo
16.
Talanta ; 235: 122814, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517670

RESUMO

Simple and sensitive detection of telomerase activity is of vital importance for both early diagnosis and therapy of malignant tumors. Inspired by DNA-biobarcode amplification reported by Chad A. Mirkin, we developed a facile DNA-biobarcode-like SERS-based copper-mediated signal amplification strategy for sensitive detection of telomerase activity. In this strategy, a duplex DNA constructed by hybridization of a copper oxide nanoparticle (CuO NP)-labeled reporting sequence (RS) with the telomerase primer sequence (TS) is ingeniously designed, and anchored on the magnetic bead (MB) to build the CuO NPs-encoded magnetic bead (MB-CuO NPs) detection probe. Upon selective sensing of telomerase, telomerase elongation reaction and structure change of TS products make the CuO NP-RS displace and separate from MB. The separated CuO NPs are dissolved into a mass of Cu2+, which prompt monodisperse dopamine-functionalized AgNPs (D-AgNPs) signal probe into aggregation, resulting in color changes and significantly enhancing of SERS signal. The SERS signal increases with the increase of Cu2+, which is directly proportional to the telomerase. Benefiting from the transformation of CuO NP to Cu2+ with a high amplification effect, this strategy could realize the telomerase activity measurement down to 3 HeLa cells and a dynamic range of 10-10000 cells. It shows a significant improvement of sensitivity without need for other enzymes and elaborate design, which escapes from the complicated manipulations and design in polymerase chain reaction (PCR) and DNA amplification techniques. Moreover, with this strategy, telomerase activities of different cell lines and telomerase inhibitors screening were successfully performed. Significantly, it can also be utilized for visual detection of telomerase, which validates the potential on-site application and its application as point-of-care testing (POCT) for efficient monitoring. Given the high-performance for telomerase analysis, the strategy has a promising application in biological detection and clinical diagnosis, as well as point-of-care tests.


Assuntos
Técnicas Biossensoriais , Telomerase , Cobre , DNA , Células HeLa , Humanos , Técnicas de Amplificação de Ácido Nucleico , Telomerase/genética , Telomerase/metabolismo
17.
Enzyme Microb Technol ; 150: 109878, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489031

RESUMO

In this article we describe a sensitive exonuclease III detection method using a DNB nanoarray from a BGISEQ-500 sequencing kit and demonstrate a detection limit as low as 0.001 U/mL. The flow cell of the sequencing kit was loaded with billions of DNA nanoballs (DNBs) to form the DNB nanoarray and initially used for massively parallel sequencing. The 3'-end recessed dsDNA structure formed by sequencing was shown to be a perfect substrate for exonuclease III, but not for other nucleases such as exonuclease I, RecJf and nuclease P1. We developed an exonuclease III assay using the DNB nanoarray, together with other reagents within the BGISEQ-500 sequencing kit, which only required one additional cycle of sequencing. The DNB nanoarray can be reused for the exonuclease III assay at least five times. This method demonstrated superior sensitivity, selectivity, and reusability compared with other assay methods and is accompanied by low cost and simple setup.


Assuntos
DNA , Tecnologia , Exodesoxirribonucleases
18.
BMC Genomics ; 22(1): 643, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34488624

RESUMO

BACKGROUND: The lower Dipteran fungus fly, Sciara coprophila, has many unique biological features that challenge the rule of genome DNA constancy. For example, Sciara undergoes paternal chromosome elimination and maternal X chromosome nondisjunction during spermatogenesis, paternal X elimination during embryogenesis, intrachromosomal DNA amplification of DNA puff loci during larval development, and germline-limited chromosome elimination from all somatic cells. Paternal chromosome elimination in Sciara was the first observation of imprinting, though the mechanism remains a mystery. Here, we present the first draft genome sequence for Sciara coprophila to take a large step forward in addressing these features. RESULTS: We assembled the Sciara genome using PacBio, Nanopore, and Illumina sequencing. To find an optimal assembly using these datasets, we generated 44 short-read and 50 long-read assemblies. We ranked assemblies using 27 metrics assessing contiguity, gene content, and dataset concordance. The highest-ranking assemblies were scaffolded using BioNano optical maps. RNA-seq datasets from multiple life stages and both sexes facilitated genome annotation. A set of 66 metrics was used to select the first draft assembly for Sciara. Nearly half of the Sciara genome sequence was anchored into chromosomes, and all scaffolds were classified as X-linked or autosomal by coverage. CONCLUSIONS: We determined that X-linked genes in Sciara males undergo dosage compensation. An entire bacterial genome from the Rickettsia genus, a group known to be endosymbionts in insects, was co-assembled with the Sciara genome, opening the possibility that Rickettsia may function in sex determination in Sciara. Finally, the signal level of the PacBio and Nanopore data support the presence of cytosine and adenine modifications in the Sciara genome, consistent with a possible role in imprinting.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Cromossomo X , DNA , Feminino , Fungos , Humanos , Masculino , Análise de Sequência de DNA
19.
Acta Cytol ; 65(5): 385-392, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34482310

RESUMO

OBJECTIVE: The objective of this study was to evaluate the application of DNA ploidy analysis in large-scale population screening for cervical cancer. METHODS: From March 2016 to March 2019, eligible subjects were enrolled and recommended to undergo DNA ploidy analysis, the ThinPrep cytology test (TCT), and high-risk human papillomavirus (hrHPV) detection concurrently. Patients with positive results were recommended for colposcopy, and biopsy diagnosis was regarded as the "gold standard." We compared the test efficiencies of the 3 methods and compared the efficiency and accuracy of the TCT in our hospital and the "2-cancer screening" project in Hubei Province during the same period. RESULTS: Among 20,574 women, the positive rates of DNA ploidy analysis, cytology, and hrHPV testing were 4.01%, 4.71%, and 16.28%, respectively. The sensitivities of these methods for screening for grade 2+ cervical intraepithelial neoplasia were 0.70, 0.68, and 0.96, and their specificities were 0.79, 0.82, and 0.45, respectively. On comparing DNA ploidy analysis with the TCT, there was no significant difference in the sensitivity, specificity, positive predictive value, negative predictive value, and missed diagnosis rate. In opportunistic screening and the 2-cancer screening project, the positive rates of cytology were 4.71% and 2.87%, respectively. And the efficiency and accuracy of the TCT in opportunistic screening were higher than in the 2-cancer screening project. CONCLUSION: Therefore, DNA ploidy analysis, which is of low-cost and does not depend on cytopathologists, can replace cytology and be applied in large-scale population screening for cervical cancer.


Assuntos
Neoplasia Intraepitelial Cervical/genética , Programas de Rastreamento , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/genética , Adulto , Biópsia , Neoplasia Intraepitelial Cervical/diagnóstico , Neoplasia Intraepitelial Cervical/patologia , Citodiagnóstico/métodos , DNA , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Papillomaviridae/genética , Gravidez , Sensibilidade e Especificidade , Neoplasias do Colo do Útero/patologia , Adulto Jovem
20.
J Acoust Soc Am ; 150(1): 241, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34340483

RESUMO

Double-strand breaks (DSBs) of giant DNA molecules after exposure to 1.0 MHz pulsed-wave ultrasound were quantitatively evaluated by single-molecule observation of giant DNA (T4 GT7 DNA; 166 kbp) through fluorescence microscopy. Aqueous solutions of DNA were exposed to ultrasonic waves with different sound pressures, repetition periods (1, 2, 5 ms), and pulse durations (5, 10, 50 µs). Below a threshold value of sound pressure, almost no double-strand breaks were generated, and above the threshold, the degree of damage increased in an accelerated manner as the pressure increased. DNA damage was much more severe for exposure to ultrasound with a shorter pulse duration. In addition, a longer pulse repetition period caused worse damage in DNA molecules. The effect of microbubbles on the damage induced by exposure to ultrasound had also been studied. While a result showed that a very small amount of microbubbles increased DSBs of DNA, this effect of microbubbles only weakly depended on their concentrations.


Assuntos
DNA , Ondas Ultrassônicas , Microbolhas , Ultrassonografia
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