RESUMO
End resection in homologous recombination (HR) and HR-mediated repair of DNA double-strand breaks (DSBs) removes several kilobases from 5' strands of DSBs, but 3' strands are exempted from degradation. The mechanism by which the 3' overhangs are protected has not been determined. Here, we established that the protection of 3' overhangs is achieved through the transient formation of RNA-DNA hybrids. The DNA strand in the hybrids is the 3' ssDNA overhang, while the RNA strand is newly synthesized. RNA polymerase III (RNAPIII) is responsible for synthesizing the RNA strand. Furthermore, RNAPIII is actively recruited to DSBs by the MRN complex. CtIP and MRN nuclease activity is required for initiating the RNAPIII-mediated RNA synthesis at DSBs. A reduced level of RNAPIII suppressed HR, and genetic loss > 30 bp increased at DSBs. Thus, RNAPIII is an essential HR factor, and the RNA-DNA hybrid is an essential repair intermediate for protecting the 3' overhangs in DSB repair.
Assuntos
RNA Polimerase III/metabolismo , Reparo de DNA por Recombinação , Ciclo Celular , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Células HEK293 , Humanos , Proteína Homóloga a MRE11/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Hibridização de Ácido Nucleico , RNA/químicaRESUMO
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously producing new variants, necessitating effective therapeutics. Patients are not only confronted by the immediate symptoms of infection but also by the long-term health issues linked to long COVID-19. Activation of epidermal growth factor receptor (EGFR) signalling during SARS-CoV-2 infection promotes virus propagation, mucus hyperproduction, and pulmonary fibrosis, and suppresses the host's antiviral response. Over the long term, EGFR activation in COVID-19, particularly in COVID-19-induced pulmonary fibrosis, may be linked to the development of lung cancer. In this review, we have summarised the significance of EGFR signalling in the context of SARS-CoV-2 infection. We also discussed the targeting of EGFR signalling as a promising strategy for COVID-19 treatment and highlighted erlotinib as a superior option among EGFR inhibitors. Erlotinib effectively blocks EGFR and AAK1, thereby preventing SARS-CoV-2 replication, reducing mucus hyperproduction, TNF-α expression, and enhancing the host's antiviral response. Nevertheless, to evaluate the antiviral efficacy of erlotinib, relevant clinical trials involving an appropriate patient population should be designed.
Assuntos
COVID-19 , Receptores ErbB , Transdução de Sinais , Humanos , Antivirais/uso terapêutico , Antivirais/farmacologia , Tratamento Farmacológico da COVID-19 , Receptores ErbB/genética , Receptores ErbB/metabolismo , Cloridrato de Erlotinib/uso terapêutico , Síndrome de COVID-19 Pós-Aguda , Fibrose Pulmonar/metabolismo , SARS-CoV-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Defects in laterality pattern can result in abnormal positioning of the internal organs during the early stages of embryogenesis, as manifested in heterotaxy syndrome and situs inversus, while laterality defects account for 3~7% of all congenital heart defects (CHDs). However, the pathogenic mechanism underlying most laterality defects remains unknown. In this study, we recruited 70 laterality defect patients with CHDs to identify candidate disease genes by exome sequencing. We then evaluated rare, loss-of-function (LOF) variants, identifying candidates by referring to previous literature. We chose TRIP11, DNHD1, CFAP74, and EGR4 as candidates from 776 LOF variants that met the initial screening criteria. After the variants-to-gene mapping, we performed function research on these candidate genes. The expression patterns and functions of these four candidate genes were studied by whole-mount in situ hybridization, gene knockdown, and gene rescue methods in zebrafish models. Among the four genes, trip11, dnhd1, and cfap74 morphant zebrafish displayed abnormalities in both cardiac looping and expression patterns of early signaling molecules, suggesting that these genes play important roles in the establishment of laterality patterns. Furthermore, we performed immunostaining and high-speed cilia video microscopy to investigate Kupffer's vesicle organogenesis and ciliogenesis of morphant zebrafish. Impairments of Kupffer's vesicle organogenesis or ciliogenesis were found in trip11, dnhd1, and cfap74 morphant zebrafish, which revealed the possible pathogenic mechanism of their LOF variants in laterality defects. These results highlight the importance of rare, LOF variants in identifying disease-related genes and identifying new roles for TRIP11, DNHD1, and CFAP74 in left-right patterning. Additionally, these findings are consistent with the complex genetics of laterality defects.
Assuntos
Cardiopatias Congênitas , Síndrome de Heterotaxia , Animais , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Padronização Corporal/genética , Cardiopatias Congênitas/metabolismo , Síndrome de Heterotaxia/genética , Síndrome de Heterotaxia/metabolismo , Cílios/genética , Cílios/metabolismoRESUMO
Bimodal-type multiplexed immunoassays with complementary mode-based correlation analysis are gaining increasing attention for enhancing the practicability of the lateral flow immunoassay (LFIA). Nonetheless, the restriction in visually indistinguishable multitargets induced by a single fluorescent color and difficulty in single acceptor ineffectual fluorescence quenching due to the various spectra of multiple different donors impede the further execution of colorimetric-fluorescence bimodal-type multiplexed LFIAs. Herein, the precise spectral overlap-based donor-acceptor pair construction strategy is proposed by regulating the size of the nanocore, coating it with an appropriate nanoshell, and selecting a suitable fluorescence donor with distinct colors. By in situ coating Prussian blue nanoparticles (PBNPs) on AuNPs with a tunable size and absorption spectrum, the resultant APNPs demonstrate efficient fluorescence quenching ability, higher colloidal stability, remarkable colorimetric intensity, and an enhanced antibody coupling efficiency, all of which facilitate highly sensitive bimodal-type LFIA analysis. Following integration with competitive-type immunoreaction, this precise spectral overlap-supported spatial separation traffic light-typed colorimetric-fluorescence dual-response assay (coined as the STCFD assay) with the limits of detection of 0.013 and 0.152 ng mL-1 for ractopamine and clenbuterol, respectively, was proposed. This work illustrates the superiority of the rational design of a precise spectral overlap-based donor-acceptor pair, hinting at the enormous potential of the STCFD assay in the point-of-care field.
Assuntos
Clembuterol , Nanopartículas Metálicas , Ouro , Imunoensaio , Fenômenos Químicos , Limite de DetecçãoRESUMO
Flexible composite polymer electrolytes (CPEs) with inorganic electrolyte fillers dispersed in polymer electrolytes integrate the merits of the polymer and inorganic electrolytes and have attracted much attention in recent years. In order to increase the electrochemical performance, especially the low lithium (Li)-ion transference number in traditional dual-ion Li salt-containing CPEs, single-ion conductive CPEs are synthesized with a single-ion polymer conductor (SIPC) as the matrix and Li6.4 La3 Zr1.4 Ta0.6 O12 (LLZTO) particles as the active fillers. The single-ion conductive CPEs show a high Li-ion transference number (up to 0.96), high room-temperature (RT) ionic conductivity (>1.0 × 10-4 S cm-1 ), wide electrochemical stability window (>5.0 V, vs Li/Li+ ), and excellent long-term cycling stability with Li metal at RT (3200 h). Based on the SIPC-LLZTO CPE, the solid-state lithium metal batteries with LiFePO4 - and LiCoO2 -based cathodes deliver average discharge capacities of 159 mAh g-1 for 600 cycles and 119 mAh g-1 for 200 cycles at RT, respectively. This study sheds light on the design of high-performance CPEs for next-generation solid-state lithium metal batteries.
RESUMO
Circular RNA is an important regulator for non-small cell lung cancer (NSCLC). Circ_0000735 has been found to be significantly overexpressed in NSCLC tissues. Therefore, its role and mechanism in NSCLC progression need to be further explored. The expression levels of circ_0000735, miR-345-5p and A disintegrin and metalloprotease 19 (ADAM19) were determined using quantitative real-time PCR. EdU staining, wound healing and transwell assays were utilized to detect cell proliferation and metastasis. The protein levels of metastasis markers, exosome markers and ADAM19 were determined using western blot. Animal experiments were performed to confirm the role of circ_0000735 in NSCLC tumorigenesis. The exosomes from cells and serum were identified using transmission electron microscopy and nanoparticle tracking analysis. We found that circ_0000735 was upregulated in NSCLC, and its knockdown repressed NSCLC cell proliferation and metastasis. In terms of mechanism, circ_0000735 targeted miR-345-5p to regulate ADAM19. MiR-345-5p inhibitor reversed the suppressive effect of circ_0000735 knockdown on NSCLC progression, and ADAM19 overexpression abolished the inhibition effect of miR-345-5p on NSCLC progression. Also, animal experiments showed that silencing of circ_0000735 reduced NSCLC tumorigenesis. In addition, exosomes mediated the intercellular transmission of circ_0000735, and serum exosomal circ_0000735 might be an important indicator for the diagnosis of NSCLC. In conclusion, circ_0000735 facilitated NSCLC progression via miR-345-5p/ADAM19 pathway, and serum exosomal circ_0000735 might be a potential biomarker for NSCLC diagnosis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Animais , Neoplasias Pulmonares/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinogênese , Transformação Celular Neoplásica , Proliferação de Células , MicroRNAs/genéticaRESUMO
Municipal solid waste incineration (MSWI) fly ash contains large amounts of Ca, Si, and other elements, giving it the potential to be used as a raw material for cement production. However, fly ash often contains a high content of salts, which greatly limits its blending ratio during cement production. These salts are commonly removed via water washing, but this process is affected by the nature and characteristics of fly ash. To clarify the influence of the ash characteristics on salt removal, a total of 60 fly ash samples from 13 incineration plants were collected, characterized, and washed. The ash characterization and cluster analysis showed that the incinerator type and flue gas purification technology/process significantly influenced the ash characteristics. Washing removed a high percentage of salts from fly ash, but the removal efficiencies varied significantly from each other, with the chlorine removal efficiency ranging from 73.76% to 96.48%, while the sulfate removal efficiency ranged from 6.92% to 51.47%. Significance analysis further revealed that the salt removal efficiency varied not only between the ash samples from different incinerators, but also between samples collected at different times from the same incinerator. The high variance of the 60 ash samples during salt removal was primarily ascribed to their different mineralogical and chemical characteristics. Mineralogical analysis of the raw and washed ash samples showed that the mineralogical forms and proportion of these salts in each ash sample greatly influenced their removal. The presence of less-soluble and insoluble chloride salts (e.g., CaClOH, Ca2Al(OH)6(H2O)2Cl etc.) in fly ash significantly affected the chlorine removal efficiency. This study also found that Fe, Mn, and Al in fly ash were negatively correlated with the dechlorination efficiency of fly ash. In summary, the different physical and chemical properties of fly ash caused great discrepancies in salt removal. Consequently, it is suggested to consider the potential impact of the ash source and ash generation time on salt removal to ensure a reliable treatment efficiency for engineering applications.
Assuntos
Cinza de Carvão , Incineração , Resíduos Sólidos , Cinza de Carvão/química , China , Resíduos Sólidos/análise , Sais/químicaRESUMO
CYP2A7 is one of the most understudied human cytochrome P450 enzymes and its contributions to either drug metabolism or endogenous biosynthesis pathways are not understood, as its only known enzymatic activities are the conversions of two proluciferin probe substrates. In addition, the CYP2A7 gene contains four single-nucleotide polymorphisms (SNPs) that cause missense mutations and have minor allele frequencies (MAFs) above 0.5. This means that the resulting amino acid changes occur in the majority of humans. In a previous study, we employed the reference standard sequence (called CYP2A7*1 in P450 nomenclature). For the present study, we created another CYP2A7 sequence that contains all four amino acid changes (Cys311, Glu169, Gly479, and Arg274) and labeled it CYP2A7-WT. Thus, it was the aim of this study to identify new substrates and inhibitors of CYP2A7 and to compare the properties of CYP2A7-WT with CYP2A7*1. We found several new proluciferin probe substrates for both enzyme variants (we also performed in silico studies to understand the activity difference between CYP2A7-WT and CYP2A7*1 on specific substrates), and we show that while they do not act on the standard CYP2A6 substrates nicotine, coumarin, or 7-ethoxycoumarin, both can hydroxylate diclofenac (as can CYP2A6). Moreover, we found ketoconazole, 1-benzylimidazole, and letrozole to be CYP2A7 inhibitors.
Assuntos
Hidrocarboneto de Aril Hidroxilases , Inibidores Enzimáticos , Humanos , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Polimorfismo de Nucleotídeo Único , Especificidade por SubstratoRESUMO
Developing signal tracers (STAs) with large size, multifunctionality, and high retention bioaffinity is believed to be a potential solution for achieving high-performance immunochromatographic assays (ICAs). However, the size limitations of STAs on strips are always a challenge because of the serious steric hindrance. Here, based on metal-quinone coordination and further metal etching, hollow micron-tubular STAs formed by natural alizarin and Fe3+ ions (named ALIFe) are produced to break through size limitations, provide more active sites, and achieve three-mode ICAs (ALIFe STAs-ICAs). Thanks to the special tubular morphology, ALIFe can successfully pass through the strip and provide an ideal signal intensity within 7 min at low mAb and probe dosages to achieve stable ICA analysis. Importantly, ALIFe shows excellent antibody enrichment and bioaffinity retention capability. With a proof-of-concept for streptomycin, the ALIFe STAs-ICAs showed the limit of detection (LOD) at 0.39 ng mL-1 for colorimetric mode, 0.32 ng mL-1 for catalytic mode, and 0.016 ng mL-1 for photothermal mode with total recoveries ranging from 80.46 to 121.59% in mike and honey samples. We anticipate that our study will help expand the ideas for the design of high-performance STAs with large size and broaden the practical application of ICA.
Assuntos
Antibacterianos , Nanopartículas Metálicas , Cromatografia de Afinidade/métodos , Limite de Detecção , Nanopartículas Metálicas/químicaRESUMO
It is of great importance to overcome potential incompatibility problems between dyestuffs and antibodies (mAbs) for extensive commercial application of a dyestuff-chemistry-based ultrafast colorimetric lateral flow immunoassay (cLFIA). Herein, inspired by traditional staining technologies, a basic dyestuff gallocyanine (GC)-assisted biogenic "potential scalpel"-based cLFIA (GC-ABPS-based cLFIA) by employing clenbuterol (CLE) as proof-of-concept was proposed to solve a high degree of incompatibility between the same potential dyestuffs and mAbs. Goat antimouse immunoglobulin (Ab2) could serve as the "potential scalpel" to form the positive potential value biomolecular network self-assemblers (BNSA) with anti-CLE mAbs (AbCLE) by noncovalent force. The cLFIA completed the entire detection process from de novo to detection results within 30 min thanks to the easy availability and ideal marking efficiency (≤1 min, saving 0.4-10 h) of GC. Encouragingly, the proposed ultrafast GC-ABPS-based cLFIA has also exhibited high sensitivity (0.411 ng mL-1) and low cost (300 times) compared with other cLFIAs. Also, the feasibility of the proposed cLFIA was demonstrated by detecting CLE in beef, pork ham, and skim milk. Finally, the proposed GC-ABPS-based cLFIA has broadened the application range of dyestuffs and provided an effective reference strategy for the application of dyestuffs in food safety monitoring.
Assuntos
Clembuterol , Animais , Bovinos , Imunoensaio/métodos , Inocuidade dos Alimentos , Anticorpos MonoclonaisRESUMO
Expanding sensing modes and improving catalytic performance of nanozyme-based analytical chemistry are beneficial to realizing the desired biosensing of analytes. Herein, Schiff-base chemistry coupled with a novel catechol oxidase-like nanozyme (CHzyme) is designed and constructed, exhibiting two main advantages, including (1) improving catalytic performance by nearly 2-fold compared with only the oxidase-like role of CHzyme; (2) increasing the designability of the output signal by signal transduction of cascade reaction. Thereafter, the substrate sensing modes based on a cascade reaction between the CHzyme-catalyzed reaction and Schiff-base chemistry are proposed and comprehensively studied, containing catalytic substrate sensing mode, competitive substrate sensing mode, and generated substrate sensing mode, expecting to be employed in environmental monitoring, food analyses, and clinical diagnoses, respectively. More meaningfully, the generated substrate sensing mode is successfully applied to construct a cascade reaction coupling ratiometric fluorescent immunoassay for the detection of clenbuterol, increasing 15-fold in detection sensitivity compared with the traditional enzyme-linked immunosorbent assay. It is expected that the expanded universal substrate sensing modes and the Schiff-base chemistry-enhanced nanozyme can enlighten the exploration of innovative biosensors.
Assuntos
Técnicas Biossensoriais , Catecol Oxidase , Ensaio de Imunoadsorção EnzimáticaRESUMO
Engineered collaborative size regulation and shape engineering of multi-functional nanomaterials (NPs) offer extraordinary opportunities for improving the analysis performance. It is anticipated to address the difficulty in distinguishing color changes caused by subtle variations in target concentrations, thereby facilitating the highly sensitive analysis of lateral flow immunoassays (LFIAs). Herein, tremella-like gold-manganese oxide (Au-MnOx ) nanoparticles with precise MnCl2 regulation are synthesized as immuno signal tracers via a facile one-step redox reaction in alkaline condition at ambient temperature. Avail of the tunable elemental composition and anisotropy in morphology, black-colored tremella-like Au-MnOx exhibits superb colorimetric signal brightness, enhanced antibody coupling efficiency, marvelous photothermal performance, and unrestricted immunological recognition affinity, all of which facilitate highly sensitive multi-signal transduction patterns. In conjunction with the handheld thermal reader device, a bimodal-type LFIA that combines size-regulation- and shape-engineering-mediated colorimetric-photothermal dual-response assay (coined as the SSCPD assay) with a limit of detection of 0.012 ng mL-1 for ractopamine (RAC) monitoring is achieved by integrating Au-MnOx with the competitive-type immunoreaction. This work illustrates the effectiveness of this strategy for establishing high-performance sensing, and the SSCPD assay may be extended to a wide spectrum of future point-of-care (POC) diagnostic applications.
Assuntos
Nanopartículas Metálicas , Nanopartículas , Ouro , Imunoensaio , Anticorpos , Colorimetria , Limite de DetecçãoRESUMO
Within the past 2 decades, three highly pathogenic human coronaviruses have emerged, namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The health threats and economic burden posed by these tremendously severe coronaviruses have paved the way for research on their etiology, pathogenesis, and treatment. Compared to SARS-CoV and SARS-CoV-2, MERS-CoV genome encoded fewer accessory proteins, among which the ORF4b protein had anti-immunity ability in both the cytoplasm and nucleus. Our work for the first time revealed that ORF4b protein was unstable in the host cells and could be degraded by the ubiquitin proteasome system. After extensive screenings, it was found that UBR5 (ubiquitin protein ligase E3 component N-recognin 5), a member of the HECT E3 ubiquitin ligases, specifically regulated the ubiquitination and degradation of ORF4b. Similar to ORF4b, UBR5 can also translocate into the nucleus through its nuclear localization signal, enabling it to regulate ORF4b stability in both the cytoplasm and nucleus. Through further experiments, lysine 36 was identified as the ubiquitination site on the ORF4b protein, and this residue was highly conserved in various MERS-CoV strains isolated from different regions. When UBR5 was knocked down, the ability of ORF4b to suppress innate immunity was enhanced and MERS-CoV replication was stronger. As an anti-MERS-CoV host protein, UBR5 targets and degrades ORF4b protein through the ubiquitin proteasome system, thereby attenuating the anti-immunity ability of ORF4b and ultimately inhibiting MERS-CoV immune escape, which is a novel antagonistic mechanism of the host against MERS-CoV infection. IMPORTANCE ORF4b was an accessory protein unique to MERS-CoV and was not present in SARS-CoV and SARS-CoV-2 which can also cause severe respiratory disease. Moreover, ORF4b inhibited the production of antiviral cytokines in both the cytoplasm and the nucleus, which was likely to be associated with the high lethality of MERS-CoV. However, whether the host proteins regulate the function of ORF4b is unknown. Our study first determined that UBR5, a host E3 ligase, was a potential host anti-MERS-CoV protein that could reduce the protein level of ORF4b and diminish its anti-immunity ability by inducing ubiquitination and degradation. Based on the discovery of ORF4b-UBR5, a critical molecular target, further increasing the degradation of ORF4b caused by UBR5 could provide a new strategy for the clinical development of drugs for MERS-CoV.
Assuntos
Infecções por Coronavirus , Interações entre Hospedeiro e Microrganismos , Coronavírus da Síndrome Respiratória do Oriente Médio , Proteólise , Ubiquitina-Proteína Ligases , Ubiquitinação , Proteínas Virais , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/virologia , Citocinas/imunologia , Humanos , Imunidade Inata , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Terapia de Alvo Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , SARS-CoV-2 , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinas/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação ViralRESUMO
2-Alkylquinolones are a class of microbial natural products primarily produced in the Pseudomonas and Burkholderia genera that play a key role in modulating quorum sensing. Bacterial alkylquinolones were synthesized and then subjected to oxidative biotransformation using human cytochrome P450 enzyme CYP4F11, heterologously expressed in the fission yeast Schizosaccharomyces pombe. This yielded a range of hydroxylated and carboxylic acid derivatives which had undergone ω-oxidation of the 2-alkyl chain, the structures of which were determined by analysis of NMR and MS data. Oxidation efficiency depended on chain length, with a chain length of eight or nine carbon atoms proving optimal for high yields. Homology modeling suggested that Glu233 was relevant for binding, due to the formation of a hydrogen bond from the quinolone nitrogen to Glu233, and in this position only the longer alkyl chains could come close enough to the heme moiety for effective oxidation. In addition to the direct oxidation products, a number of esters were also isolated, which was attributed to the action of endogenous yeast enzymes on the newly formed ω-hydroxy-alkylquinolones. ω-Oxidation of the alkyl chain significantly reduced the antimicrobial and antibiofilm activity of the quinolones.
Assuntos
Bactérias , Sistema Enzimático do Citocromo P-450 , Humanos , Oxirredução , Sistema Enzimático do Citocromo P-450/metabolismo , Família 4 do Citocromo P450/metabolismoRESUMO
Ferroptosis, characterized by glutamate overload, glutathione depletion, and cysteine/cystine deprivation during iron- and oxidative-damage-dependent cell death, is a particular mode of regulated cell death. It is expected to effectively treat cancer through its tumor-suppressor function, as mitochondria are the intracellular energy factory and a binding site of reactive oxygen species production, closely related to ferroptosis. This review summarizes relevant research on the mechanisms of ferroptosis, highlights mitochondria's role in it, and collects and classifies the inducers of ferroptosis. A deeper understanding of the relationship between ferroptosis and mitochondrial function may provide new strategies for tumor treatment and drug development based on ferroptosis.
Assuntos
Ferroptose , Neoplasias , Humanos , Morte Celular , Ferro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Peroxidação de LipídeosRESUMO
The assay of kinase activity with ultrahigh sensitivity is important to medical diagnostics and drug discovery. Herein, we report the biologically mediated RAFT polymerization (BMRP) and its potential use as an efficient amplification strategy in the ultrasensitive electrochemical sensing of kinase activity. In BMRP, the reversible addition-fragmentation chain-transfer (RAFT) process is initiated and sustained by the reduced form of coenzyme I (i.e., NADH), which can efficiently mediate the direct fragmentation of thiocarbonylthio (TCT) compounds (or the TCT-capped dormant chains) to produce an initiating/propagating radical under mild conditions. Due to the absence of exogenous radicals, the notorious radical termination in RAFT equilibrium can be greatly suppressed. For the sensing of kinase activity, the recognition peptides, without carboxyl groups, are immobilized via the Au-S self-assembly. After phosphorylation, TCT compounds (as RAFT agents) are tethered to the enzymatically generated phosphate groups via the carboxylate-Zr(IV)-phosphate (CZP) linkage. Subsequently, the BMRP of ferrocenylmethyl methacrylate (FcMMA) results in the labeling of each phosphate group with hundreds to thousands of Fc tags, thereby greatly amplifying the sensing signal. Obviously, the BMRP-based strategy is biologically friendly, highly efficient, uncomplicated, and quite low-cost. The detection limit of 1.85 mU/mL has been achieved toward the selective sensing of the cAMP-dependent protein kinase (PKA). Moreover, the proposed kinase sensor is applicable to inhibitor screening and kinase activity sensing in serum samples. By virtue of its low cost, high sensitivity and selectivity, and uncomplicated operation, the proposed kinase sensor holds great potential in medical diagnostics and drug discovery.
Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Fosfatos , Fosforilação , PolimerizaçãoRESUMO
Multiplex lateral flow immunoassay (mLFIA) has attracted great attention due to the increasing need for rapid detection of multiple analytes. However, it has a number of disadvantages with regard to accuracy and interference because of difficulties in simplifying the process of preparing nanomaterial-based probes. In this work, inspired by protein self-assembly, for the first time, a facile natural antibody network (NAN)-based mLFIA for multiple chloramphenicol (CAP) and streptomycin (STR) determination was designed. The NAN structure was constructed by introducing a second antibody (Ab2) as a scaffold to noncovalently combine with various monoclonal antibodies (mAbs), thus permitting each mAb to act as an independent functional unit to maintain bioactivity. Furthermore, the NAN was colored by simple one-step staining using coomassie brilliant blue R-250 (CBBR) to form a chromogenic probe, eliminating the need for complex nanomaterials to improve reproducibility and precision. Under optimal conditions, a satisfactory detection performance (the visual limit of detection (v-LOD) of 3 ng mL-1 for CAP and 20 ng mL-1 for STR) was obtained for whole milk analysis, which met the basic requirement of detection and had good specificity, reproducibility (relative standard deviation (RSD) < 15%), and robustness. In addition, the precision of the detection results was improved usefully since the test procedure was simplified. Overall, the developed system enables fast, simple, and reliable point-of-care assays of multiple analytes.
Assuntos
Leite , Testes Imediatos , Animais , Imunoensaio/métodos , Limite de Detecção , Leite/química , Reprodutibilidade dos TestesRESUMO
As a class of oligosaccharide chain-containing proteins, glycoproteins are of great value in screening and early diagnosis of malignant tumors and other major diseases. Herein, we report a universal boronate affinity-based electrochemical aptasensor for point-of-care glycoprotein detection. Aptasensing of glycoproteins involves the specific recognition and capture of target glycoproteins by end-tethered nucleic acid aptamers and the site-specific labeling of ferrocene tags via the phenylboronic acid (PBA)-based boronate affinity interactions because the cis-diol sites of oligosaccharide chains on glycoproteins can selectively react with the PBA receptor groups to form cyclic phenylborates in aqueous basic media. Due to the presence of hundreds to thousands of cis-diol sites on a glycoprotein, a large number of ferrocene tags can be recruited for the signal-on aptasensing of glycoproteins at a low-abundance level, eliminating the need for extra amplification strategies. As a result, the boronate affinity-based electrochemical aptasensor is highly sensitive and selective for glycoprotein detection and tolerant to the false-positive results. The detection limit for α-fetoprotein (AFP) is 0.037 ng/mL, with a linear response ranging from 0.1 to 100 ng/mL. In addition to the merits of simple operation, short assay time, and low detection cost, the aptasensor is applicable to the detection of glycoproteins in serum samples and the point-of-care detection using disposable flexible electrodes. Overall, this work provides a universal and promising platform for the point-of-care detection of glycoproteins, holding great potential in screening and early diagnosis of glycoprotein-related malignant tumors and other major diseases.
Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Eletrodos , Glicoproteínas , Ouro , Limite de Detecção , Metalocenos , Sistemas Automatizados de Assistência Junto ao LeitoRESUMO
Stimulated surface-enhanced Raman scattering (SERS) in combination with engineered nano-tracer offers extraordinary potential in lateral flow immunoassays (LFIAs). Nonetheless, the investigation execution of SERS-LFIA is often compromised by the intricacy and overlap of the Raman fingerprint spectrum as well as the affinity-interference of nano-tracer to antibody. To circumvent these critical issues, an engineered core-shell multifunctional nano-tracer (named APNPs) with precise control of the size of nano-core (AuNPs) and coating of the nano-shell (Prussian blue nanomaterials) is prepared for SERS-LFIA via a modified enlarging particle size and coating modification strategy. Importantly, this nano-tracer exhibits enhanced coupling efficiency, highly retained affinity, reinforced colloid stability, and unique SERS signal (2156 cm-1 ) in the silent region (1800-2800 cm-1 ) with high signal-to-background ratio simultaneously, all of which are beneficial to the enhancement of the analysis performance. With a proof-of-concept demonstration for detection of ractopamine (RAC), a dual-pattern LFIA that synergizes both the enlarged particle size and coating modification supported colorimetric/biological silence Raman dual-response (coined as the ECCRD assay) is demonstrated by integrating APNPs with the competitive-type immunoreaction. This research may contribute to the rational design of multifunctional nano-tracer, and the ECCRD assay can be expanded for a wide spectrum of applications in environmental monitoring and biomedical diagnosis.
Assuntos
Ouro , Nanopartículas Metálicas , Prata , Análise Espectral Raman , ImunoensaioRESUMO
In vivo proton magnetic resonance spectroscopy (1H-MRS) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are two semi-quantitative analytical methods commonly used in neurochemical research. In this study, the two methods were used complementarily, in parallel, to investigate neurochemical perturbations in the medial prefrontal cortex (mPFC) of 9-month-old DJ-1 knockout mice, a well-established transgenic model for Parkinson's diseases. Convergingly, the results obtained with the two methods demonstrated that, compared with the wild-type (WT) mice, the DJ-1 knockout mice had significantly increased glutathione (GSH) level and GSH/glutamate (Glu) ratio in the mPFC, which likely presented an astrocytic compensatory mechanism in response to elevated regional oxidative stress induced by the loss of DJ-1 function. The results from this study also highlighted (1) the need to be cautious when interpreting the in vivo 1H-MRS results obtained from aged transgenic animals, in which the concentration of internal reference, being whether water or total creatine, could no longer be assumed to be the same as that in the age-matched WT animals, and (2) the necessity and importance of complementary analyses with more than one method under such circumstances.