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Mutation in oncogene KRas plays a crucial role in the occurrence and progression of numerous malignant tumors. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. We hypothesize that oncogene KRas mutations are intrinsic to alterations in cellular mechanics that promote malignant tumor generation and progression. Here, we demonstrate the use of optical tweezers coupled with a confocal fluorescence imaging system and gene interference technique to reveal that the mutant KRas protein can be transported between homogeneous and heterogeneous tumor cells by tunneling nanotubes (TNTs), resulting in a significant reduction of membrane tension and acceleration of membrane phospholipid flow in the recipient cells. Simultaneously, the changes in membrane mechanical properties of the tumor cells also enhance the metastatic and invasive ability of the tumors, which further contribute to the deterioration of the tumors. This finding helps to clarify the association between oncogene mutations and changes in the mechanical properties of tumor cells, which provides a theoretical basis for the development of cancer treatment strategies. STATEMENT OF SIGNIFICANCE: Here, we present a laser confocal fluorescence system integrated with optical tweezers to observe the transfer of mutant KRasG12D protein from mutant cells to wild-type cells through TNTs. Malignancy involves changes in cell mechanics for extensive cellular deformation during metastatic dissemination. Our results demonstrate a significant decrease in membrane tension and an increase in membrane phospholipid flow in recipient cells. These alterations in mechanical properties augment the migration and invasive capabilities of tumor cells, contributing to tumor malignancy. Our findings propose that cellular mechanical properties could serve as new markers for tumor development, and targeting membrane tension may hold potential as a therapeutic strategy.
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Viral diseases are among the main threats to public health. Understanding the factors affecting viral invasion is important for antiviral research. Until now, it was known that most viruses have very low plaque-forming unit (PFU)-to-particle ratios. However, further investigation is required to determine the underlying factors. Here, using quantitative single-particle analysis methods, the invasion of Semliki Forest virus (SFV), Japanese encephalitis virus (JEV), and influenza A virus (IAV) containing attachment to the cell surface, entry into the cell, transport towards the cell interior, and fusion with endosomes to release nucleocapsids were quantitatively analysed in parallel. It was found that for SFV with an PFU-to-particle ratio of approximately 1:2, an entry efficiency of approximately 31% limited infection. For JEV, whose PFU-to-particle ratio was approximately 1:310, an attachment efficiency of approximately 27% and an entry efficiency of 10% were the main factors limiting its infection. Meanwhile, for IAV with PFU-to-particle ratios of 1:8100, 5% attachment efficiency, 9% entry efficiency, and 53% fusion efficiency significantly limited its infection. These results suggest that viruses with different infectivities have different limited steps in the invasion process. Moreover, there are significant differences in attachment efficiencies among viruses, emphasizing the pivotal role of attachment in viral invasion. The influence of the virus purification method on virus invasion was also investigated. This study, for the first time, reports the efficiencies of different stages of virus invasion, leading to a better understanding of virus invasion and providing a protocol to quantitatively analyse the virus invasion efficiency.
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Vírus da Influenza A , Vírus da Floresta de Semliki , Internalização do Vírus , Vírus da Influenza A/fisiologia , Animais , Vírus da Floresta de Semliki/fisiologia , Humanos , Vírus da Encefalite Japonesa (Espécie)/fisiologia , Linhagem Celular , Ligação Viral , Endossomos/virologiaRESUMO
The CRISPR-Cas system has been found to be extremely sensitive and there is an urgent demand to extend its potential in bioassays. Herein, we developed a novel nanobiosensor to detect the human papillomavirus 16 genes (HPV-16 DNA), which is triggered by CRISPR-Cas12a to amplify the fluorescence signal by metal-enhanced fluorescence (CAMEF). Along with the changing of the fluorescence signal, the aggregation of the substrate of MEF also leads to a change in the color of the mixture solution, enabling dual signal detection with the fluorescence and the naked eye. Furthermore, the designed CAMEF probe was verified to detect the HPV-16 DNA accurately and reliably in biological samples. Triggered by the CRISPR system, the designed CAMEF probe allows quantitative detection of the HPV-16 DNA in the wide range of 10-500 pM. Owing to the MEF, the fluorescence signal of the CAMEF probe was significantly amplified with the detection limit as low as 1 pM. Besides, we can determine the concentration of HPV-16 DNA simply by the naked eye, which also drastically reduces the possibility of false-positive signals. Theoretically, the target ssDNA could be any strand of DNA obtained by designing the crRNA sequence in the CRISPR-Cas system. We believe that the designed CAMEF sensor can present a reliable approach for the accurate detection of low amounts of target ssDNA in complex biological samples.
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Técnicas Biossensoriais , Sistemas CRISPR-Cas , Colorimetria , DNA Viral , Papillomavirus Humano 16 , Sistemas CRISPR-Cas/genética , Papillomavirus Humano 16/genética , Colorimetria/métodos , Humanos , DNA Viral/análise , DNA Viral/genética , Técnicas Biossensoriais/métodos , Limite de Detecção , Fluorescência , Corantes Fluorescentes/química , Espectrometria de Fluorescência/métodosRESUMO
While there is significant potential for DNA machine-built enzyme-free fluorescence biosensors in the imaging analysis of live biological samples, they persist certain shortcomings. These encompass a deficiency of signal enrichment within a singular interface, uncontrolled premature activation during bio-delivery, and a slow reaction rate due to free nucleic acid collisions. In this contribution, we are committed to resolving the above challenges. Firstly, a single-interface-integrated domino-like driving amplification is constructed. In this conception, a specific target acts as the domino promotor (namely the energy source), initiating a cascading chain reaction that grafts onto a singular interface. Next, an 808 nm near-infrared (NIR) light-excited up-converting luminescence-induced light-activatable biosensing technique is introduced. By locking the target-specific identification segment with a photo-cleavage connector, the up-converted ultraviolet emission can activate target binding in a completely controlled manner. Moreover, a fast reaction rate is achieved by confining nucleic acid collisions within the surface of a DNA wire nano-scaffold, leading to a substantial enhancement in local contact concentration (30.8-fold increase, alongside a 15 times elevation in rate). When a non-coding microRNA (miRNA-221) is positioned as the model low-abundance target for proof-of-concept validation, our intelligent DNA machine demonstrates ultra-high sensitivity (with a limit of detection down to 62.65 fM) and good specificity for this hepatic malignant tumor-associated biomarker in solution detection. Going further, it is worth highlighting that the biosensing system can be employed to carry out high-performance imaging analysis in live bio-samples (ranging from the cellular level to the nude mouse body), thereby propelling the field of DNA machines in disease diagnosis.
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Técnicas Biossensoriais , DNA , Raios Infravermelhos , MicroRNAs , Técnicas Biossensoriais/métodos , Humanos , DNA/química , DNA/genética , MicroRNAs/análise , MicroRNAs/genética , Animais , Camundongos , Técnicas de Amplificação de Ácido Nucleico/métodos , Imagem Óptica/métodos , Nanoestruturas/químicaRESUMO
While DNA amplifier-built nanobiosensors featuring a DNA polymerase-free catalytic hairpin assembly (CHA) reaction have shown promise in fluorescence imaging assays within live biosystems, challenges persist due to unsatisfactory precision stemming from premature activation, insufficient sensitivity arising from low reaction kinetics, and poor biostability caused by endonuclease degradation. In this research, we aim to tackle these issues. One aspect involves inserting an analyte-binding unit with a photoinduced cleavage bond to enable a light-powered notion. By utilizing 808 nm near-infrared (NIR) light-excited upconversion luminescence as the ultraviolet source, we achieve entirely a controllable sensing event during the biodelivery phase. Another aspect refers to confining the CHA reaction within the finite space of a DNA self-assembled nanocage. Besides the accelerated kinetics (up to 10-fold enhancement) resulting from the nucleic acid restriction behavior, the DNA nanocage further provides a 3D rigid skeleton to reinforce enzymatic resistance. After selecting a short noncoding microRNA (miRNA-21) as the modeled low-abundance sensing analyte, we have verified that the innovative NIR light-powered and DNA nanocage-confined CHA nanobiosensor possesses remarkably high sensitivity and specificity. More importantly, our sensing system demonstrates a robust imaging capability for this cancer-related universal biomarker in live cells and tumor-bearing mouse bodies, showcasing its potential applications in disease analysis.
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Técnicas Biossensoriais , DNA , Raios Infravermelhos , MicroRNAs , MicroRNAs/análise , Humanos , Técnicas Biossensoriais/métodos , Animais , DNA/química , Camundongos , Imagem Óptica , Nanoestruturas/químicaRESUMO
Correction for 'A label-free and immobilization-free approach for constructing photoelectrochemical nucleic acid sensors utilizing DNA-silver nanoparticle affinity interactions' by Jing Yi et al., Analyst, 2024, https://doi.org/10.1039/D4AN00098F.
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Efficient and affordable nucleic acid detection methods play a pivotal role in various applications. Herein, we developed an immobilization-free and label-free strategy to construct a photoelectrochemical nucleic acid biosensing platform based on interactions between silver nanoparticles and DNA. First, CRISPR-Cas12a exhibited a trans-cleavage effect on adenine nucleotide sequences upon recognizing the target DNA. The resulting adenine nucleotide sequences of varying lengths then engaged in interactions with silver nanoparticles, leading to a solution characterized by distinct light transmittance. Subsequently, the solution was positioned between the light source and the photoelectrode, strategically impacting the photon absorption step within the photoelectrochemical process. Consequently, the detection of nucleic acid was accomplished through the analysis of the resultant photocurrent signal. The developed platform exhibits a detection limit of 0.06 nM (S/N = 3) with commendable selectivity. The innovative use of adenine nucleotide sequences as cost-effective probes interacting with silver nanoparticles eliminates the need for complex interfacial immobilization processes, significantly simplifying the fabrication of DNA sensors. The outcomes of our research present a promising pathway for advancing the development of economically feasible miniature DNA sensors.
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Técnicas Biossensoriais , Nanopartículas Metálicas , Ácidos Nucleicos , Hibridização de Ácido Nucleico/métodos , Nanopartículas Metálicas/química , Prata/química , Técnicas Biossensoriais/métodos , DNA/química , AdeninaRESUMO
While three-dimensional (3D) DNA walking amplifiers hold considerable promise in the construction of advanced DNA-based fluorescent biosensors for bioimaging, they encounter certain difficulties such as inadequate sensitivity, premature activation, the need for exogenous propelling forces, and low reaction rates. In this contribution, a variety of profitable solutions have been explored. First, a catalytic hairpin assembly (CHA)-achieved nonenzymatic isothermal nucleic acid amplification is integrated to enhance sensitivity. Subsequently, one DNA component is simply functionalized with a photocleavage-bond to conduct a photoresponsive manner, whereby the target recognition occurs only when the biosensor is exposed to an external ultraviolet light source, overcoming premature activation during biodelivery. Furthermore, a special self-propelling walking mechanism is implemented by reducing biothiols to MnO2 nanosheets, thereby propelling forces that are self-supplied to a Mn2+-reliant DNAzyme. By carrying the biosensing system with a DNA molecular framework to induce a unique concentration localization effect, the nucleic acid contact reaction rate is notably elevated by 6 times. Following these, an ultrasensitive in vitro detection performance with a limit of detection down to 2.89 fM is verified for a cancer-correlated microRNA biomarker (miRNA-21). Of particular importance, our multiple concepts combined 3D DNA walking amplifier that enables highly efficient fluorescence bioimaging in live cells and even bodies, exhibiting a favorable application prospect in disease analysis.
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Técnicas Biossensoriais , DNA Catalítico , MicroRNAs , DNA Catalítico/química , Compostos de Manganês , Óxidos , DNA/química , MicroRNAs/análise , Técnicas Biossensoriais/métodos , Limite de DetecçãoRESUMO
Multiple coexisting seasonal lakes are observed in the Poyang Lake basin. The interaction between surface water and groundwater, along with solute transport at the sediment-water interface (SWI), plays a crucial role in material cycling within the Poyang Lake ecosystem. However, the mechanisms governing how the relative positions of these lakes influence solute transport at the SWI remain unclear. This study employs indoor experiments and simulations based on real topography to investigate how the separation distance and elevation differences between two seasonal lakes, termed "lake A" (situated farther from the main lake) and "lake B" (closer to the main lake), affect solute transport. Findings highlight a distinct recharge pattern from lake A to lake B and the main lake during periodic water level fluctuations. A reduced distance between dual seasonal lakes results in a diminished water level drop in lake B during dry seasons. Proximity allows lake A to contribute more solutes to the main lake while promoting solute transport from lake B to the main lake, increasing the pore water recharge flux to overlying water in lake B. In cases where the separation distance has insufficient impact on water levels, the speed of pore water flow in this area inversely correlates with the distance between dual lakes. Reducing the distance intensifies solute transport into the bottom of lake A. Lower the elevation of lake B increases the water level difference between dual seasonal lakes, curtailing pollution within the lakebed. Elevating lake B forms hydrological isolation and more severe pollution of the lakebed. Solutes predominantly transport between lake B and the main lake, with pollution spreading to the lakebed of lake A and transitioning to downward diffusion over time. This research provides valuable insights for the hydraulic regulation of seasonal lakes and holds significance for the ecological restoration of Poyang Lake.
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Although nucleic acids-based fluorescent biosensors, exemplified by the hybridization chain reaction (HCR), have exhibited promise as an imaging tool for detecting disease-related biomolecular makers in living biosystems, they still face certain challenges. These include the need for improved sensitivity, poor bio-targeting capability, the absence of signal enrichment interface and the uncontrollable biosensing initiation. Herein, we present a range of effective solutions. First, a stacking design resembling building blocks is used to construct a special hierarchical HCR (termed H-HCR), for which a hierarchical bridge is employed to graft multiunit HCR products. Furthermore, the H-HCR components are encapsulated into a virus-like particle (VLP) endowed with a naturally peptide-mediated targeting unit through genetic engineering of plasmids, after which the biosensor can specifically identify cancer cytomembranes. By further creating a multibranched DNA scaffold to enrich the H-HCR produced detection signals, the biosensor's analyte recognition module is inserted with a photocleavage-linker, allowing that the biosensing process can be spatiotemporally initiated via a light-powered behavior. Following these innovations, this genetically engineered VLP-armoured and multibranched DNA-scaffold-corbelled H-HCR demonstrates an ultra-sensitive and specific biosensing performance to a cancer-associated microRNA marker (miRNA-155). Beyond the worthy in vitro analysis, our method is also effective in performing imaging assays for such low-abundance analyte in living cells and even bodies, thus providing a roust platform for disease diagnosis.
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Técnicas Biossensoriais , MicroRNAs , Neoplasias , Humanos , Técnicas Biossensoriais/métodos , Hibridização de Ácido Nucleico/métodos , DNA/genética , DNA/análise , MicroRNAs/genéticaRESUMO
Optical tweezers use the momentum of photons to capture and manipulate particles in a noncontact way. Although related techniques have been widely used in biology and materials, research on viruses is still relatively limited. It is hard to optically trap viruses because trap stiffness is rather low and the size of viruses is too small. Here, we used an optical tweezers system coupled with a laser confocal fluorescence imaging system, which allows individual viruses to be imaged and trapped in real time and analyzed using multiple parameters in the culture medium. We show that a single virus tagged by quantum dots (QDs) can increase the real part of polarizability, further increasing gradient force and trap stiffness. With this method, we not only can trap and manipulate viruses in real time but also can analyze their interactions with other targets.
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Dispositivos Ópticos , Pontos Quânticos , Pinças Ópticas , Fótons , Movimento (Física)RESUMO
In recent years, optical tweezers have become a novel tool for biodetection, and to improve the inefficiency of a single trap, the development of multitraps is required. Herein, we constructed a set of hybrid multitrap optical tweezers with the balance of stability and flexibility by the combination of two different beam splitters, a diffraction optical element (DOE) and galvano mirrors (GMs), to capture polystyrene (PS) microbeads in aqueous solutions to create an 18-trap suspended array. A sandwich hybridization strategy of DNA-miRNA-DNA was adopted to detect three kinds of target miRNAs associated with triple negative breast cancer (TNBC), in which different upconversion nanoparticles (UCNPs) with red, green, and blue emissions were applied as luminescent tags to encode the carrier PS microbeads to further indicate the levels of the targets. With encoded luminescent microbeads imaged by a three-channel microscopic system, the biodetection displayed high sensitivity with low limits of detection (LODs) of 0.27, 0.32, and 0.33 fM and exceptional linear ranges of 0.5 fM to 1 nM, 0.7 fM to 1 nM, and 1 fM to 1 nM for miR-343-3p, miR-155, and miR-199a-5p, respectively. In addition, this bead-based assay method was demonstrated to have the potential for being applied in patients' serum by satisfactory standard addition recovery experiment results.
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MicroRNAs , Humanos , MicroRNAs/genética , Microesferas , Pinças Ópticas , PoliestirenosRESUMO
Extreme hydrological events have become increasingly frequent on a global scale. The middle Yangtze River also faces a substantial challenge in dealing with extreme flooding and drought. However, the long-term characteristics of the extreme hydrological regime have not yet been adequately recognized. Moreover, there is uncertainty in the extreme value estimation, and this uncertainty needs to be distinguished and quantified. In this study, we investigated the nonstationary frequency characteristics of extreme low lake levels (ELLLs), taking the Poyang Lake as an example. Daily lake levels from 1960 to 2022 were utilized to estimate the return level using the generalized Pareto distribution (GPD). The uncertainty from three sources, i.e., the parameter estimator, threshold selection, and covariate, was quantified via variance decomposition. The results indicate that (1) the parameter estimator is the predominant source of uncertainty, with a contribution rate of approximately 87 %. The total uncertainty of the covariate, threshold, and interaction term is only 13 %. (2) Two indexes, namely the annual minimum water level (WLmin) and the days with peak over the 90 % threshold per year (DPOT90), decreased (0.01-0.03 m/year) and increased (0.17-1.39 days/year), respectively, indicating a progressively severe drought trend for Poyang Lake. (3) The return level with return period of 5 to 100 years significantly decreased after the early 21st century. A large spatial heterogeneity was identified for the variation in the return level, and the change rate of the return level with a 100-year return period ranged from 5 % to 40 % for the whole lake. (4) The ELLLs had a stronger correlation with the catchment discharge than with the Yangtze River discharge and the large-scale atmospheric circulation indices. This study provides a methodology with reduced uncertainty for nonstationary frequency analysis (NFA) of ELLLs exemplified in large river-lake systems.
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The detection of hydrogen sulfide (H2S), the third gas signaling molecule, is a promising strategy for identifying the occurrence of certain diseases. However, the conventional single- or dual-signal detection can introduce false-positive or false-negative results, which ultimately decreases the diagnostic accuracy. To address this limitation, we developed a luminescent, photothermal, and electrochemical triple-signal detection platform by optically trapping the synthetic highly doped upconversion coupled SiO2 microbeads coated with metal-organic frameworks H-UCNP-SiO2@HKUST-1 (H-USH) to detect the concentration of H2S. The H-USH was first synthesized and proved to have stable structure and excellent luminescent, photothermal, and electrochemical properties. Under 980 nm optical trapping and 808 nm irradiation, H-USH showed great detection linearity, a low limit of detection, and high specificity for H2S quantification via triple-signal detection. Moreover, H-USH was captured by optical tweezers to realize quantitative detection of H2S content in serum of acute pancreatitis and spontaneously hypertensive rats. Finally, by analyzing the receiver operating characteristic (ROC) curve, we concluded that triple-signal detection of H2S was more accurate than single- or dual-signal detection, which overcame the problem of false-negative/positive results in the detection of H2S in actual serum samples.
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Sulfeto de Hidrogênio , Pancreatite , Ratos , Animais , Sulfeto de Hidrogênio/química , Luminescência , Eletroquímica , Doença Aguda , Dióxido de Silício , MicroesferasRESUMO
Achieving facile control of the wavelength of light emitters is of great significance for many key applications in optoelectronics and photonics, including on-chip interconnection, super-resolution imaging, and optical communication. The Joule heating effect caused by electric current is widely applied in modulating the refractive index of silicon-based waveguides for reconfigurable nanophotonic circuits. Here, by utilizing localized Joule heating in the biased graphene device, we demonstrate electrically controlled wavelength-tunable photoluminescence (PL) from vertical van der Waals heterostructures combined by graphene and two-dimensional transition metal dichalcogenides (2D-TMDCs). By applying a moderate electric field of 6.5 kV·cm-1 to the graphene substrate, the PL wavelength of 2D-TMDCs exhibits a continuous tuning from 662 to 690 nm, corresponding to a bandgap reduction of 76 meV. The electric control is highly reversible during sweeping the bias back and forth. The temperature dependence of Raman and PL spectroscopy reveals that the current-induced local Joule heating effect plays a leading role in reducing the optical direct bandgap of TMDCs. The bias-dependent optical reflectivity and time-resolved photoluminescence measurements show a consistent reduction of the optical band gap of 2D-TMDCs and increased PL lifetimes with the electric field over the heterostructures. Moreover, we demonstrate the consistent device operation from 2D materials grown by chemical vapor deposition, showing great advantages for the scalability.
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Although great headway has been made in DNAzyme-based detection of Pb2+, its adaptability, sensitivity, and accessibility in complex media still need to be improved. For this, we introduce new ways to surmount these hurdles. First, a spherical nucleic acid (SNA) fluorescence probe (Au nanoparticles-DNAzyme probe) is utilized to specifically identify Pb2+ and its suitability for precise detection of Pb2+ in complex samples due to its excellent nuclease resistance. Second, the sensitivity of Pb2+ detection is greatly enhanced via the use of a clustered regularly interspaced short palindromic repeats-Cas12a with target recognition accuracy to amplify the fluorescent signal upon the trans cleavage of the SNA (signal probe), and the limit of detection reaches as low as 86 fM. Third, we boost the fluorescence on photonic crystal chips with a bionic periodic arrangement by employing a straightforward detection device (smartphone and portable UV lamp) to achieve on-site detection of Pb2+ with the limit of detection as low as 24 pM. Based on the abovementioned efforts, the modified Pb2+ fluorescence sensor has the advantages of higher sensitivity, better specificity, accessibility, less sample consumption, and so forth. Moreover, it can be applied to accurately detect Pb2+ in complex biological or environmental samples, which is of great promise for widespread applications.
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DNA Catalítico , Nanopartículas Metálicas , Sistemas CRISPR-Cas , Ouro , ChumboRESUMO
Attenuation of groundwater ammonium (NH4+) is expected to occur through redox reaction and adsorption of the riverbank. Previous studies determined that NH4+ mostly degraded through nitrification along subsurface flow, however, the adsorption capacities of riverbanks were always ignored in the NH4+ reduction processes. In this study, field experiments were conducted in the Fuliji section of the Xiaosuixin River, China, to understand NH4+ transport and attenuation under rainfall events-induced river and groundwater interactions. The results indicated that the NH4+ concentration in river water increased significantly after heavy rainfall events and reached a peak of about 5.88 mg L-1, and the lag time was more than 2 weeks compared with the river peak stage. Adsorption plays a dominant role in attenuation of NH4+ in riverbank with high amounts of organic materials and clay minerals, reducing its concentration to less than 0.05 mg L-1. A two-dimensional lateral exchange and transport model of NH4+ was developed and calibrated against observations in the aquifer, and an exponential reduction pattern of NH4+ was identified. The model's possible implications about the effects of varying hydrologic changes (i.e., peak stage and lag time differences between river and groundwater) on NH4+ transport were also discussed. Thus, the effects of river-groundwater interactions on nitrogen pollution should be taken into consideration in river regulation strategies in order to ensure proper hydrogeochemical functioning of river-aquifer interfaces and related ecosystems.
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Compostos de Amônio , Água Subterrânea , Poluentes Químicos da Água , China , Ecossistema , Hidrologia , Rios , Poluentes Químicos da Água/análiseRESUMO
Influenza A virus (IAV) is a global health threat. The cellular endocytic machineries harnessed by IAV remain elusive. Here, by tracking single IAV particles and quantifying the internalized IAV, we found that sphingomyelin (SM)-sequestered cholesterol, but not accessible cholesterol, is essential for the clathrin-mediated endocytosis (CME) of IAV. The clathrin-independent endocytosis of IAV is cholesterol independent, whereas the CME of transferrin depends on SM-sequestered cholesterol and accessible cholesterol. Furthermore, three-color single-virus tracking and electron microscopy showed that the SM-cholesterol complex nanodomain is recruited to the IAV-containing clathrin-coated structure (CCS) and facilitates neck constriction of the IAV-containing CCS. Meanwhile, formin-binding protein 17 (FBP17), a membrane-bending protein that activates actin nucleation, is recruited to the IAV-CCS complex in a manner dependent on the SM-cholesterol complex. We propose that the SM-cholesterol nanodomain at the neck of the CCS recruits FBP17 to induce neck constriction by activating actin assembly. These results unequivocally show the physiological importance of the SM-cholesterol complex in IAV entry. IMPORTANCE IAV infects cells by harnessing cellular endocytic machineries. A better understanding of the cellular machineries used for its entry might lead to the development of antiviral strategies and would also provide important insights into physiological endocytic processes. This work demonstrated that a special pool of cholesterol in the plasma membrane, SM-sequestered cholesterol, recruits FBP17 for the constriction of clathrin-coated pits in IAV entry. Meanwhile, the clathrin-independent cell entry of IAV is cholesterol independent. The internalization of transferrin, the gold-standard cargo endocytosed solely via CME, is much less dependent on the SM-cholesterol complex. These results provide new insights into IAV infection and the pathway/cargo-specific involvement of the cholesterol pool(s).
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Colesterol , Vesículas Revestidas por Clatrina , Proteínas de Ligação a Ácido Graxo , Forminas , Vírus da Influenza A , Internalização do Vírus , Actinas/metabolismo , Animais , Colesterol/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Vesículas Revestidas por Clatrina/virologia , Endocitose/fisiologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Forminas/metabolismo , Vírus da Influenza A/metabolismo , Domínios Proteicos , Esfingomielinas/metabolismo , Transferrinas/metabolismoRESUMO
The early diagnosis and timely intervention of viral myocarditis urgently require a noninvasive detection approach. Therefore, we present a CRISPR/Cas12a-powered biosensor that integrates an exceptionally efficient upconversion luminescent resonance energy transfer (LRET) with a nature-inspired biochip to determine a golden-standard cardiac biomarker (cardiac troponin I). First, a unique sandwich-structured energy-confined upconversion nanoparticle (acting as the energy donor) is synthesized to dramatically reinforce the LRET's ability. Such a structural improvement endows a relatively high quenching efficiency (as much as 93.8%) toward the surface acceptors and enhances the working adaption in complicated biological media. Moreover, a three-dimensional photonic crystal fabricated using a self-assembly of nanospheres is employed to construct a biochip interface, under which the upconversion luminescence is prominently boosted to approximately 27-fold to achieve signal amplification. Finally, the newly developed luminescence sensing method exhibits remarkable assay performance after introducing these attempts into a dual-aptamer-regulated CRISPR/Cas12a system to transduce the target. More importantly, this biosensor can primarily be a quite useful tracer tool to allow dynamic monitoring of the entire myocardial injury process in a coxsackievirus B3 infected mouse model, paving an attractive venue for medical diagnostic techniques.
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Técnicas Biossensoriais , Miocardite , Nanosferas , Animais , Sistemas CRISPR-Cas , Transferência Ressonante de Energia de Fluorescência , Camundongos , Miocardite/diagnósticoRESUMO
The further development of high-performance fluorescent biosensors to image intracellular microRNAs is beneficial to cancer medicine. By virtue of the need for enzymes and hairpin DNA probes, the entropy-driven reaction-assisted signal amplification strategy has shown an enormous potential to accomplish this task. Nevertheless, this good option still meets with poor biostability, low cell uptake efficiency, and unsatisfactory accuracy. On the basis of these challenges, we put forward here a battery of solving pathways. First, the straight DNA probes are anchored onto the vertexes of dual DNA tetrahedrons, and thus the enzyme resistance of the whole sensing system is observably enhanced. A metal-organic framework (ZIF-8 nanoparticle), which can be effectively dissociated into a weakly acidic environment, then is employed as an additional delivery vehicle to encapsulate such a DNA tetrahedron sustained biosensor and finally bring about a more efficient endocytosis. Last, a kind of photocleavage-linker triggered photoresponsive manner is incorporated to achieve an exceptional precise target identification, by which the biosensor can only be initiated under the irradiation of an externally mild 365 nm ultraviolet light source. In accordance with the above efforts, worthy assay performance toward microRNA-196a has given rise to this newly constructed biosensor, whose sensitivity is down to 2.7 pM and also able to distinguish single-base variation. Beyond that, the amplifier can work as a powerful imaging toolbox to accurately determine the targets in living cells, providing a promising intracellular sensing platform.