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Effective detection of infectious pathogens is crucial for disease prevention and control. We present an innovative Internet of Things (IoT) molecular diagnostic device featuring a WeChat mini-program for simultaneous detection and spatiotemporal mapping of respiratory pathogens. Leveraging social software's widespread usage, our device integrates seamlessly with WeChat, eliminating the need for app downloads and installations. Through a comprehensive detection system, including a user-friendly mini-program, a portable Point-of-Care fluorescence detector, and a diagnostic information management platform (EzDx Cloud), we demonstrate high sensitivity and specificity in detecting common respiratory viruses. Our SARS-CoV-2/H1N1 combo test kit, developed using a novel one-tube/one-step loop-mediated isothermal amplification-CRISPR method, shows remarkable performance. We address challenges in at-home nucleic acid testing by providing a cost-effective solution capable of detecting multiple pathogens simultaneously. Our system's versatility accommodates various assays operating at different temperatures and fluorescence intensities, offering significant advantages over traditional methods. Moreover, integration with EzDx Cloud facilitates disease monitoring and early warning systems, enhancing public health management. This study highlights the potential of our IoT molecular diagnostic device in revolutionizing infectious disease detection and control, with wide-ranging applications in both human and animal population.
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A novel bromothiophene-functionalized BF2-curcuminoid (BTC-BF2) is synthesized by Knoevenagel condensation reaction. The structure of BTC-BF2 is determined by 1H-nuclear magnetic resonance (1H NMR), 13C-nuclear magnetic resonance (13C NMR), and high-resolution mass spectrometry (HRMS). Moreover, a nearly coplanar single crystal structure is successfully obtained and form a mesh structure through intermolecular multiple CâH···F hydrogen bond interactions. As expected, as-prepared BTC-BF2 exhibits solvent-dependent photophysical properties in solvents with different polarity and an intense red solid-state fluorescence. Density functional theory calculations further verify the relationships between its intrinsic electronic features and the photophysical properties. For its potential application aspect, BTC-BF2 shows a certain ability to generate singlet oxygen under irradiation with 530 nm green light. Moreover, BTC-BF2 can be utilized as versatile building block to construct novel far-red or NIR BF2-curcuminoid complexes for widely biological applications.
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Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and â¼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (â¼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.
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Proteínas Argonautas/genética , Ácidos Nucleicos Livres/genética , Neoplasias/genética , Ácidos Nucleicos Peptídicos/genética , Alelos , Humanos , Mutação/genética , Neoplasias/diagnóstico , Neoplasias/patologia , Ácidos Nucleicos Peptídicos/isolamento & purificação , Thermus thermophilus/genéticaRESUMO
Liquid biopsy is a highly promising method for non-invasive detection of tumor-associated nucleic acid fragments in body fluids but is challenged by the low abundance of nucleic acids of clinical interest and their sequence homology with the vast background of nucleic acids from healthy cells. Recently, programmable endonucleases such as clustered regularly interspaced short palindromic repeat (CRISPR) associated protein (Cas) and prokaryotic Argonautes have been successfully used to remove background nucleic acids and enrich mutant allele fractions, enabling their detection with deep next generation sequencing (NGS). However, the enrichment level achievable with these assays is limited by futile binding events and off-target cleavage. To overcome these shortcomings, we conceived a new assay (Programmable Enzyme-Assisted Selective Exponential Amplification, PASEA) that combines the cleavage of wild type alleles with concurrent polymerase amplification. While PASEA increases the numbers of both wild type and mutant alleles, the numbers of mutant alleles increase at much greater rates, allowing PASEA to achieve an unprecedented level of selective enrichment of targeted alleles. By combining CRISPR-Cas9 based cleavage with recombinase polymerase amplification, we converted samples with 0.01% somatic mutant allele fractions (MAFs) to products with 70% MAFs in a single step within 20 min, enabling inexpensive, rapid genotyping with such as Sanger sequencers. Furthermore, PASEA's extraordinary efficiency facilitates sensitive real-time detection of somatic mutant alleles at the point of care with custom designed Exo-RPA probes. Real-time PASEA' performance was proved equivalent to clinical amplification refractory mutation system (ARMS)-PCR and NGS when testing over hundred cancer patients' samples. This strategy has the potential to reduce the cost and time of cancer screening and genotyping, and to enable targeted therapies in resource-limited settings.
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Short of a vaccine, frequent and rapid testing, preferably at home, is the most effective strategy to contain the COVID-19 pandemic. Herein, we report on single-stage and two-stage molecular diagnostic tests that can be carried out with simple or no instrumentation. Our single-stage amplification is reverse transcription-loop mediated isothermal amplification (RT-LAMP) with custom-designed primers targeting the ORF1ab and the N gene regions of the virus genome. Our new two-stage amplification, dubbed Penn-RAMP, comprises recombinase isothermal amplification (RT-RPA) as its first stage and LAMP as its second stage. We compared various sample preparation strategies aimed at deactivating the virus while preserving its RNA and tested contrived and patient samples, consisting of nasopharyngeal swabs, oropharyngeal swabs, and saliva. Amplicons were detected either in real time with fluorescent intercalating dye or after amplification with the intercalating colorimetric dye LCV, which is insensitive to sample's PH. Our single RT-LAMP tests can be carried out instrumentation-free. To enable concurrent testing of multiple samples, we developed an inexpensive heat block that supports both the single-stage and two-stage amplification. Our RT-LAMP and Penn-RAMP assays have, respectively, analytical sensitivities of 50 and 5 virions/reaction. Both our single- and two-stage assays have successfully detected SARS-CoV-2 in patients with viral loads corresponding to the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) threshold cycle smaller than 32 while operating with minimally processed samples, without nucleic acid isolation. Penn-RAMP provides a 10-fold better sensitivity than RT-LAMP and does not need thermal cycling like PCR assays. All reagents are amenable to dry, refrigeration-free storage. The SARS-CoV-2 test described herein is suitable for screening at home, at the point of need, and in resource-poor settings.
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COVID-19 , SARS-CoV-2 , Teste para COVID-19 , Humanos , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Pandemias , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
To enable inexpensive molecular detection at the point-of-care and at home with minimal or no instrumentation, it is necessary to streamline unit operations and store reagents refrigeration-free. To address this need, a multifunctional enzymatic amplification reactor that combines solid-phase nucleic acid extraction, concentration, and purification; refrigeration-free storage of reagents with just-in-time release; and enzymatic amplification is designed, prototyped, and tested. A nucleic acid isolation membrane is placed at the reactor's inlet, and paraffin-encapsulated reagents are prestored within the reactor. When a sample mixed with chaotropic agents is filtered through the nucleic acid isolation membrane, the membrane binds nucleic acids from the sample. Importantly, the sample volume is decoupled from the reaction volume, enabling the use of relatively large sample volumes for high sensitivity. When the amplification reactor's temperature increases to its operating level, the paraffin encapsulating the reagents melts and moves out of the way. The reagents are hydrated, just-in-time, and the polymerase reaction proceeds. The amplification process can be monitored, in real-time. We demonstrate our reactors' ability to amplify both DNA and RNA targets using polymerase with both reverse-transcriptase and strand displacement activities to obtain sensitivities on-par with benchtop equipment and a shelf life exceeding 6 months.
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Técnicas Analíticas Microfluídicas/instrumentação , Ácidos Nucleicos/análise , Reação em Cadeia da Polimerase/instrumentação , Extração em Fase Sólida/instrumentação , DNA Viral/análise , DNA Viral/genética , Desenho de Equipamento , Liofilização , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/isolamento & purificação , Humanos , Indicadores e Reagentes , Ácidos Nucleicos/genética , Infecções por Papillomavirus/virologia , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Rapid and quantitative molecular diagnostics in the field, at home, and at remote clinics is essential for evidence-based disease management, control, and prevention. Conventional molecular diagnostics requires extensive sample preparation, relatively sophisticated instruments, and trained personnel, restricting its use to centralized laboratories. To overcome these limitations, we designed a simple, inexpensive, hand-held, smartphone-based mobile detection platform, dubbed "smart-connected cup" (SCC), for rapid, connected, and quantitative molecular diagnostics. Our platform combines bioluminescent assay in real-time and loop-mediated isothermal amplification (BART-LAMP) technology with smartphone-based detection, eliminating the need for an excitation source and optical filters that are essential in fluorescent-based detection. The incubation heating for the isothermal amplification is provided, electricity-free, with an exothermic chemical reaction, and incubation temperature is regulated with a phase change material. A custom Android App was developed for bioluminescent signal monitoring and analysis, target quantification, data sharing, and spatiotemporal mapping of disease. SCC's utility is demonstrated by quantitative detection of Zika virus (ZIKV) in urine and saliva and HIV in blood within 45 min. We demonstrate SCC's connectivity for disease spatiotemporal mapping with a custom-designed website. Such a smart- and connected-diagnostic system does not require any lab facilities and is suitable for use at home, in the field, in the clinic, and particularly in resource-limited settings in the context of Internet of Medical Things (IoMT).
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HIV/isolamento & purificação , Imagem Óptica , Patologia Molecular , Smartphone , Zika virus/isolamento & purificação , HIV/genética , Humanos , Internet , Medições Luminescentes , Técnicas de Amplificação de Ácido Nucleico , Zika virus/genéticaRESUMO
BACKGROUND: The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS: To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP). RAMP's first-stage uses outer loop-mediated isothermal amplification (LAMP) primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent dye and a smartphone. RESULTS: In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed (<40 min); ease of use; and ability to cope with minimally processed samples. CONCLUSIONS: RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.
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Técnicas Analíticas Microfluídicas , Reação em Cadeia da Polimerase Multiplex , Técnicas de Amplificação de Ácido Nucleico , Sistemas Automatizados de Assistência Junto ao Leito , Temperatura , HumanosRESUMO
The recent outbreak of Zika virus (ZIKV) infection in the Americas and its devastating impact on fetal development have prompted the World Health Organization (WHO) to declare the ZIKV pandemic as a Public Health Emergency of International Concern. Rapid and reliable diagnostics for ZIKV are vital because ZIKV-infected individuals display no symptoms or nonspecific symptoms similar to other viral infections. Because immunoassays lack adequate sensitivity and selectivity and are unable to identify active state of infection, molecular diagnostics are an effective means to detect ZIKV soon after infection and throughout pregnancy. We report on a highly sensitive reverse-transcription loop-mediated, isothermal amplification (RT-LAMP) assay for rapid detection of ZIKV and its implementation in a simple, easy-to-use, inexpensive, point-of-care (POC) disposable cassette that carries out all the unit operations from sample introduction to detection. For thermal control of the cassette, we use a chemically heated cup without a need for electrical power. Amplification products are detected with leuco crystal violet (LCV) dye by eye without a need for instrumentation. We demonstrated the utility of our POC diagnostic system by detecting ZIKV in oral samples with sensitivity of 5 plaque-forming units (PFU) in less than 40 min. Our system is particularly suitable for resource-poor settings, where centralized laboratory facilities, funds, and trained personnel are in short supply, and for use in doctors' offices, clinics, and at home.
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Sistemas Automatizados de Assistência Junto ao Leito , Zika virus/isolamento & purificação , Primers do DNA/metabolismo , Violeta Genciana/química , Humanos , Microfluídica , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/química , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Saliva/virologia , Zika virus/genética , Infecção por Zika virus/diagnóstico , Infecção por Zika virus/virologiaRESUMO
Nucleic acid amplification-based diagnostics offer rapid, sensitive, and specific means for detecting and monitoring the progression of infectious diseases. However, this method typically requires extensive sample preparation, expensive instruments, and trained personnel. All of which hinder its use in resource-limited settings, where many infectious diseases are endemic. Here, we report on a simple, inexpensive, minimally-instrumented, smart cup platform for rapid, quantitative molecular diagnostics of pathogens at the point of care. Our smart cup takes advantage of water-triggered, exothermic chemical reaction to supply heat for the nucleic acid-based, isothermal amplification. The amplification temperature is regulated with a phase-change material (PCM). The PCM maintains the amplification reactor at a constant temperature, typically, 60-65°C, when ambient temperatures range from 12 to 35°C. To eliminate the need for an optical detector and minimize cost, we use the smartphone's flashlight to excite the fluorescent dye and the phone camera to record real-time fluorescence emission during the amplification process. The smartphone can concurrently monitor multiple amplification reactors and analyze the recorded data. Our smart cup's utility was demonstrated by amplifying and quantifying herpes simplex virus type 2 (HSV-2) with LAMP assay in our custom-made microfluidic diagnostic chip. We have consistently detected as few as 100 copies of HSV-2 viral DNA per sample. Our system does not require any lab facilities and is suitable for use at home, in the field, and in the clinic, as well as in resource-poor settings, where access to sophisticated laboratories is impractical, unaffordable, or nonexistent.
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Circulating tumor cells (CTCs) are cancer cells that break away from either a primary tumor or a metastatic site and circulate in the peripheral blood as the cellular origin of metastasis. With their role as a "tumor liquid biopsy", CTCs provide convenient access to all disease sites, including that of the primary tumor and the site of fatal metastases. It is conceivable that detecting and analyzing CTCs will provide insightful information in assessing the disease status without the flaws and limitations encountered in performing conventional tumor biopsies. However, identifying CTCs in patient blood samples is technically challenging due to the extremely low abundance of CTCs among a large number of hematologic cells. To address this unmet need, there have been significant research endeavors, especially in the fields of chemistry, materials science, and bioengineering, devoted to developing CTC detection, isolation, and characterization technologies. Inspired by the nanoscale interactions observed in the tissue microenvironment, our research team at UCLA pioneered a unique concept of "NanoVelcro" cell-affinity substrates, in which CTC capture agent-coated nanostructured substrates were utilized to immobilize CTCs with high efficiency. The working mechanism of NanoVelcro cell-affinity substrates mimics that of Velcro: when the two fabric strips of a Velcro fastener are pressed together, tangling between the hairy surfaces on two strips leads to strong binding. Through continuous evolution, three generations (gens) of NanoVelcro CTC chips have been established to achieve different clinical utilities. The first-gen NanoVelcro chip, composed of a silicon nanowire substrate (SiNS) and an overlaid microfluidic chaotic mixer, was created for CTC enumeration. Side-by-side analytical validation studies using clinical blood samples suggested that the sensitivity of first-gen NanoVelcro chip outperforms that of FDA-approved CellSearch. In conjunction with the use of the laser microdissection (LMD) technique, second-gen NanoVelcro chips (i.e., NanoVelcro-LMD), based on polymer nanosubstrates, were developed for single-CTC isolation. The individually isolated CTCs can be subjected to single-CTC genotyping (e.g., Sanger sequencing and next-generation sequencing, NGS) to verify the CTC's role as tumor liquid biopsy. Created by grafting of thermoresponsive polymer brushes onto SiNS, third-gen NanoVelcro chips (i.e., Thermoresponsive NanoVelcro) have demonstrated the capture and release of CTCs at 37 and 4 °C, respectively. The temperature-dependent conformational changes of polymer brushes can effectively alter the accessibility of the capture agent on SiNS, allowing for rapid CTC purification with desired viability and molecular integrity. This Account summarizes the continuous evolution of NanoVelcro CTC assays from the emergence of the original idea all the way to their applications in cancer research. We envision that NanoVelcro CTC assays will lead the way for powerful and cost-efficient diagnostic platforms for researchers to better understand underlying disease mechanisms and for physicians to monitor real-time disease progression.
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Separação Celular/métodos , Técnicas Analíticas Microfluídicas/métodos , Nanoestruturas/química , Células Neoplásicas Circulantes/patologia , Separação Celular/instrumentação , Humanos , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
This paper describes an associated analysis method of DNA methylation for the detection of cancer using an optically amplifying cationic conjugated polymer (CCP, poly{(1,4-phenylene)-2,7-[9,9-bis(6'-N,N,N-trimethyl ammonium)-hexyl fluorene] dibromide)}. Genomic DNA is digested by methylation-sensitive restriction endonuclease, followed by PCR amplification to incorporate fluorescein-labeled dNTP. Only methylated DNA can be amplified by PCR, and the methylation level is detected through fluorescence resonance energy transfer (FRET) between CCP and fluorescein that is incorporated into the PCR product. The methylation levels of RASSF1A, OPCML, and HOXA9 promoters of 35 ovarian cancer samples and 11 normal samples were assayed. In accordance with the degree of methylation levels, they are clustered to three sections and assigned a value. Through an associated analysis, we acquired a threshold for cancer detection with a sensitivity of 85.7%. The assay takes about 20 h to obtain the detection results and shows great potential as a useful tool for diagnostic and screening of cancer.
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Metilação de DNA/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Cátions , Feminino , Humanos , Neoplasias/metabolismoRESUMO
In light of the recent worldwide scientific and technological revolution, it is imperative that urban infrastructure undergo a digital transformation in order to lower carbon emissions and support sustainable urban growth. However, to date, there is a lack of empirical research on carbon emissions based on the digital transformation of urban infrastructure. This paper uses data from 178 prefecture-level cities in China from 2005 to 2020 to study the impact of digital transformation of urban infrastructure on carbon emissions based on the "local-neighbourhood" perspective using a spatial difference-in-differences model. The results show that the digital transformation of urban infrastructure reduces the intensity of local carbon emissions while also reducing the carbon emissions of neighbouring cities, with a spatial spillover effect, and the boundary of this spatial spillover is 600 km. Mechanistic analyses suggest that digital transformation of urban infrastructure can reduce carbon emissions locally as well as in nearby areas by promoting green technological innovations. In light of this, this study has important policy implications for maximising the contribution of digital transformation of infrastructure to reducing carbon emissions.
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Carbono , Cidades , China , Carbono/análise , Humanos , Poluição do Ar/prevenção & controle , Poluição do Ar/análise , Dióxido de Carbono/análiseRESUMO
Objective: In this study, we assessed the efficacy and safety of various thrombolytic treatment protocols in patients with hyperacute cerebral infarction. Methods: Patients diagnosed with acute ischemic stroke within 6 h of symptom onset and with brain computer tomography angiography confirming the absence of major vessel stenosis or occlusion were eligible for this study. The enrolled patients were subsequently randomized into two groups: all the groups received the standard intravenous thrombolysis treatment with rt-PA (0.9 mg/kg), and the experimental group underwent sequential intra-arterial thrombolysis treatment with alteplase (0.3 mg/kg, with a maximum dose of 22 mg), administered directly into the target vessel via a microcatheter. Both groups were closely monitored for changes in their National Institutes of Health Stroke Scale (NIHSS) score, modified Rankin scale score, hemorrhage rate, all-cause mortality rate, and the rate of favorable outcomes at 90 ± 7 days. Results: Ninety-four participants were enrolled in this study, with both the control and experimental groups initiating intravenous injection of rt-PA at a median time of 29 min. For the experimental group, the median time for arterial puncture was 123 min. Baseline data for both groups were similar (P > 0.05). Hemorrhagic transformation occurred in 24.47 % (23 patients), with a lower intracranial hemorrhage rate observed in the experimental group compared to the control group (15.2 % vs 33.3 %, P < 0.05). Asymptomatic hemorrhage rates were 8.7 % for the experimental group and 12.5 % for the control group, with no hemorrhage detected in other locations. Post-treatment median NIHSS scores were lower in the experimental group than in the control group (7 vs 9, P < 0.05), but short-term NIHSS scores were similar (P > 0.05). A higher proportion of patients in the experimental group achieved favorable outcomes compared to the control group (87.0 % vs 43.8 %, P < 0.05). Conclusion: In patients with acute ischemic stroke with an onset time of ≤6 h and no major intracranial vessel occlusion, combining rt-PA intravenous thrombolysis with intra-arterial thrombolysis via a microcatheter might yield superior functional outcomes.
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The expansion of large-scale aquaculture has exacerbated the challenge of aquatic diseases, resulting in substantial economic losses annually. Currently, traditional laboratory-based diagnostic methods are time-consuming and costly, hindering on-site testing for individual farmers. We address this issue by developing a state-of-the-art handheld isothermal nucleic acid amplification device (WeD-1) capable of fluorescence tracking of reactions and integrating it with an enhanced one-pot Prokaryotic Argonaute based nucleic acid detection method, enabling duplex visual detection of aquatic pathogens. WeD-1 is portable, reusable, user-friendly, and cost-effective, offering real-time smartphone interaction and enabling real-time fluorescence observation during the reaction. The enhanced one-pot Loop-Mediated Isothermal Amplification (LAMP)-PfAgo method, incorporating paraffin-encapsulated lyophilized PfAgo protein, achieves precise target-specific cleavage, significantly enhancing multiplex nucleic acid detection. This innovation streamlines on-site testing, negating the need for specialized laboratory conditions while ensuring an aerosol-free system. With newly developed and highly sensitive LAMP primer sets, our compact WeD-1/LAMP-PfAgo nucleic acid rapid testing system exhibits remarkable sensitivity, readily detecting aquatic pathogens with naked eyes from rapidly prepared fish and shrimp samples within 40 min, even when the Ct values are as high as 34.
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Técnicas Biossensoriais , Ácidos Nucleicos , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Sensibilidade e EspecificidadeRESUMO
A new fluorescent nanoparticle (PIOT-HA) is synthesized with cationic polyester (PIOT) and anionic hyaluronic acid (HA) by electrostatic interactions in an aqueous solution. The nanoparticles (NPs) are degradable upon treatments with alkali or hyaluronidase, which exhibits better biological safety and potential application in vitro and in vivo. Through specific interactions between the HA locating on the surfaces of PIOT-HA NPs and the CD44 protein over-expressed on the MDA-MB-231 cancer cell line, PIOT-HA NPs could selectively image the cancer cells. Upon white light irradiation, the PIOT-HA NPs can sensitize oxygen to generate reactive oxygen species (ROS) that inactivate the neighboring CD44 protein, which inhibits the migration of MDA-MB-231 cancer cells.
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Corantes Fluorescentes/síntese química , Ácido Hialurônico/análogos & derivados , Ácido Hialurônico/química , Nanopartículas/química , Poliésteres/química , Técnicas Biossensoriais , Linhagem Celular Tumoral , Movimento Celular , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/síntese química , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/metabolismo , Microscopia Confocal , Rotação Ocular , Poliésteres/síntese química , Poliésteres/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
A fingerprint spectrum technique that utilizes cationic conjugated-polymer-based fluorescence resonance energy transfer (FRET) is used for multiplex detection of DNA mutations. This method detects as low as 5 % mutation of the total DNA. Ten PIK3CA mutations originating from 30 clinical breast cancer samples could be detected.
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Análise Mutacional de DNA/métodos , DNA/análise , DNA/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Mutação , Técnicas Biossensoriais , DNA/metabolismo , Impressões Digitais de DNA/métodosRESUMO
Stellera chamaejasme L. is a widely distributed poisonous plant in Chinese degraded grasslands. To investigate the role of endophytic fungi (EF) in S. chamaejasme's quick spread in grasslands, the endophytic fungal community of S. chamaejasme was studied through culture-dependent and culture-independent methods, and the plant-growth-promoting (PGP) traits of some culturable isolates were tested. Further, the growth-promoting effects of 8 isolates which showed better PGP traits were evaluated by pot experiments. The results showed that a total of 546 culturable EF were isolated from 1114 plant tissue segments, and the colonization rate (CR) of EF in roots (33.27%) was significantly higher than that in shoots (22.39%). Consistent with this, the number of specific types of EF was greater in roots (8 genera) than in shoots (1 genus). The same phenomenon was found in culture-independent study. There were 95 specific genera found in roots, while only 18 specific genera were found in shoots. In addition, the dominant EF were different between the two study methods. Cladosporium (18.13%) and Penicillium (15.93%) were the dominant EF in culture-dependent study, while Apiotrichum (13.21%) and Athelopsis (5.62%) were the dominant EF in culture-independent study. PGP trait tests indicated that 91.30% of the tested isolates (69) showed phosphorus solubilization, IAA production, or siderophores production activity. The benefit of 8 isolates on host plants' growth was further studied by pot experiments, and the results indicated that all of the isolates can improve host plants' growth. Among them, STL3G74 (Aspergillus niger) showed the best growth-promotion effect; it can increase the plant's shoot and root dry biomass by 68.44% and 74.50%, respectively, when compared with the controls. Our findings revealed that S. chamaejasme has a wide range of fungal endophytic assemblages, and most of them possess PGP activities, which may play a key role in its quick spread in degraded grasslands.