RESUMEN
BACKGROUND: Perineural invasion (PNI), as the fifth recognized pathway for the spread and metastasis of colorectal cancer (CRC), has increasingly garnered widespread attention. The preoperative identification of whether colorectal cancer (CRC) patients exhibit PNI can assist clinical practitioners in enhancing preoperative decision-making, including determining the necessity of neoadjuvant therapy and the appropriateness of surgical resection. The primary objective of this study is to construct and validate a preoperative predictive model for assessing the risk of perineural invasion (PNI) in patients diagnosed with colorectal cancer (CRC). MATERIALS AND METHODS: A total of 335 patients diagnosed with colorectal cancer (CRC) at a single medical center were subject to random allocation, with 221 individuals assigned to a training dataset and 114 to a validation dataset, maintaining a ratio of 2:1. Comprehensive preoperative clinical and pathological data were meticulously gathered for analysis. Initial exploration involved conducting univariate logistic regression analysis, with subsequent inclusion of variables demonstrating a significance level of p < 0.05 into the multivariate logistic regression analysis, aiming to ascertain independent predictive factors, all while maintaining a p-value threshold of less than 0.05. From the culmination of these factors, a nomogram was meticulously devised. Rigorous evaluation of this nomogram's precision and reliability encompassed Receiver Operating Characteristic (ROC) curve analysis, calibration curve assessment, and Decision Curve Analysis (DCA). The robustness and accuracy were further fortified through application of the bootstrap method, which entailed 1000 independent dataset samplings to perform discrimination and calibration procedures. RESULTS: The results of multivariate logistic regression analysis unveiled independent risk factors for perineural invasion (PNI) in patients diagnosed with colorectal cancer (CRC). These factors included tumor histological differentiation (grade) (OR = 0.15, 95% CI = 0.03-0.74, p = 0.02), primary tumor location (OR = 2.49, 95% CI = 1.21-5.12, p = 0.013), gross tumor type (OR = 0.42, 95% CI = 0.22-0.81, p = 0.01), N staging in CT (OR = 3.44, 95% CI = 1.74-6.80, p < 0.001), carcinoembryonic antigen (CEA) level (OR = 3.13, 95% CI = 1.60-6.13, p = 0.001), and platelet-to-lymphocyte ratio (PLR) (OR = 2.07, 95% CI = 1.08-3.96, p = 0.028).These findings formed the basis for constructing a predictive nomogram, which exhibited an impressive area under the receiver operating characteristic (ROC) curve (AUC) of 0.772 (95% CI, 0.712-0.833). The Hosmer-Lemeshow test confirmed the model's excellent fit (p = 0.47), and the calibration curve demonstrated consistent performance. Furthermore, decision curve analysis (DCA) underscored a substantial net benefit across the risk range of 13% to 85%, reaffirming the nomogram's reliability through rigorous internal validation. CONCLUSION: We have formulated a highly reliable nomogram that provides valuable assistance to clinical practitioners in preoperatively assessing the likelihood of perineural invasion (PNI) among colorectal cancer (CRC) patients. This tool holds significant potential in offering guidance for treatment strategy formulation.
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Neoplasias Colorrectales , Nomogramas , Humanos , Estudios Retrospectivos , Reproducibilidad de los Resultados , HospitalesRESUMEN
While the solid-state nanopore shows increasing potential during sensitive and label-free single molecular analysis, target concentration and signal amplification method is in urgent need. In this article, a solution via designing a model nucleic acid circuit reaction that can produce "Y" shape-structure three-way DNA oligomers with controllable size and polymerization degree is proposed. Such a so-called lego-like three-way catalytic hairpin assembly (LK-3W-CHA) can provide both concentration amplification (via CHA circuit) and programmable size control (via lego-like building mode) to enhance spatiotemporal resolution in single molecular sensing of solid-state nanopore. Oligomers containing 1-4 DNA three-way junctions (Y monomers, Y1-Y4) are designed in proof-of-concept experiments and applications. When the oligomers are applied to direct translocation measurements, Y2-Y4 can significantly increase the signal resolution and stability than that of Y1. Meanwhile, Y1 to Y4 can be used as the tags on the long DNA carrier to provide very legible secondary signals for specific identification, multiple assays, and information storage. Compared with other possible tags, Y1-Y4 provides higher signal density and amplitude, and quasi-linear "inner reference" for each other, which may provide more systematic, reliable, and controllable experimental results.
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Técnicas Biosensibles , ADN , ADN/química , Transducción de Señal , Replicación del ADN , Técnicas Biosensibles/métodos , Límite de DetecciónRESUMEN
Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target-of-interest into a specific and anti-false/inference signal, especially for solid-state nanopores with relatively lower resolution and higher noise. Here we report a high-resolution signal-production concept named target-induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target-specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono-polymerization of single DS and co-polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron-DNA structures with different sizes are used as the STs to provide needle-like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size-amplification, concentration amplification, and signal-specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.
RESUMEN
Solid-state nanopores have shown special high potential in a label-free molecular assay, structure identification, and target-index at the single-molecular level, even though frustrating electrical baseline noise is still one of the major factors that limit the spatial resolution and signaling reliability of solid-state nanopores, especially in small target detection. Here we develop a significant and easy-operating noise-reduction approach via mixing organic solvents with high dielectric constants into a traditional aqueous electrolyte. The strategy is generally effective for pores made of different materials, such as the most commonly used conical glass (CGN) or SiNx. While the mechanism should be multisourced, MD simulations suggest the noise reduction may partially arise from the even ionic distribution caused by the addition of higher dielectric species. Among all solvents experimentally tested, the two with the highest dielectric constants, formamide and methylformamide, exhibit the best noise reduction effect for target detection of CGN. The power spectral density at the low-frequency limit is reduced by nearly 3 orders with the addition of 20% formamide. Our work qualifies the reliability of solid-state nanopores into much subtler scales of detection, such as dsDNAs under 100 bp. As a practical example, bare CGN is innovatively employed to perform in-situ tracking of trigger-responsive DNA machine forming oligomers.
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ADN/química , Nanoporos , Formamidas/química , Vidrio/química , Simulación de Dinámica Molecular , NanotecnologíaRESUMEN
Recent advances have shown increasing designs of nucleic acid organizations via controlling the thermodynamics and kinetics of oligonucleotides. Nevertheless, deeper understanding and further applications of these DNA nanotechnologies are majorly hampered by the lack of effective analytical methodologies that are competent enough to investigate them. To deliver a potential solution, here we developed an innovative exploration that employed the emerging nanopore technique to characterize DNA organization at the single-molecule level and in completely homogeneous condition without covalent modification. With the help of counting and profiling the translocation-induced current drop of a DNA assembly structure passing through a conical glass nanopore (CGN), we have directly verified the formation of the individual double-helix concatemer generated from our model, hybridization chain reaction (HCR). Due to the ultrasensitivity of the nanopore technology, those concatemers that were difficult to observe on a conventional electrophoresis image were brought to light. The translocation duration time also provided the approximate length and folding information for the concatemers. These advantages were proven also applicable to structures with more sophisticated folding behaviors. Eventually, when coupling with an upstream reaction, CGN was further turned to a universal detector that was capable of even detecting other nucleic acid organization behaviors as well as targets that were unable to generate huge products. All of these results are expected to promote deeper study and applications of the nanopore technique in the field of nucleic acid nanotechnology.
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ADN Concatenado/química , Vidrio/química , Nanoporos , Oligodesoxirribonucleótidos/química , Hibridación de Ácido NucleicoRESUMEN
In the present study, a novel coordination polymer (CP) based on Ni(II), namely, [Ni(L)(D-CAM)(H2O)]n (1) (H2D-CAM = (1R,3S)-1,2,2-trimethylcyclopentane-1,3-dicarboxylic acid and L = 3,6-bis(benzimidazol-1-yl)pyridazine), has been produced successfully through applying a mixed ligand synthesis method via reacting Ni(NO3)2·6H2O with 3,6-bis(benzimidazol-1-yl)pyridazine ligand in the presence of a carboxylic acid co-ligand. Hyaluronic acid (HA) and carboxymethyl chitosan (CMCS) are representatives of natural polysaccharides and have good biocompatibility. Based on the chemical synthesis method, HA/CMCS hydrogel was successfully prepared. SEM showed that the lyophilized gel presented a typical macroporous structure with three-dimensional connected pores, which had unique advantages as a drug carrier. Using paclitaxel as a drug model, we further synthesized a novel paclitaxel-loaded metal gel and evaluated its therapeutic effect on cervical cancer. Finally, novel drugs from the reinforcement learning simulation are suggested to have better biological activity against ovarian cancer due to low affinity energy and stronger interaction strength towards the protein receptor.
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Piridazinas , Neoplasias del Cuello Uterino , Femenino , Humanos , Neoplasias del Cuello Uterino/tratamiento farmacológico , Ligandos , Hidrogeles , Ácidos Dicarboxílicos , Ácido Hialurónico , Aprendizaje Automático , Metales , PaclitaxelRESUMEN
Through controlling the ssDNA product length of rolling circle amplification with AcyNTP, here we develop a nanopore signal enhancement strategy (STSS), which can successfully transfer the short oligonucleotide targets into long ssDNAs with appropriate lengths that can generate significant translocation currents. By labelling the RCA product with tags such as tetrahedral structures and isothermal amplicons, the resolution, signal specificity, and target range of the STSS can be further extended.
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ADN de Cadena Simple , Nanoporos , Técnicas de Amplificación de Ácido Nucleico , ADN de Cadena Simple/químicaRESUMEN
BACKGROUND: The effect of radiotherapy waiting time after last induction chemotherapy (IC-RT) on prognosis of patients with locally advanced nasopharyngeal carcinoma (LANPC) needs further discussion. METHODS: Three hundred and six patients with LANPC diagnosed pathologically by induction chemotherapy (IC) and radiotherapy (RT) from 2013 to 2018 were selected for this study. RESULTS: The IC-RT was a risk factor for the post-treatment progression of LANPC (OR = 1.017 95%CI: 1.003-1.031), For patients with LANPC, the IC-RT > 40 days significantly reduced 5-year PFS (70% vs. 55%; p = 0.0012), 5-year OS (84% vs. 73%; p = 0.028), 5-year DMFS (80% vs. 66%; p = 0.003), 5-year LRFS (77% vs. 67%; p = 0.012). Indicating that patients with stage IVa who IC-RT > 40 days were found to be a significant predictor of aggravated PFS (HR = 2.69; 95%CI: 1.57-4.6), OS (HR = 2.55; 95%CI: 1.29-5.03), DMFS (HR = 3.07; 95%CI: 1.64-5.76) and LRFS (HR = 2.26; 95%CI: 1.21-4.21). CONCLUSION: The prognosis of patients will be adversely affected if the IC-RT exceeds 40 days, especially for stage IVa patients.
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Carcinoma , Neoplasias Nasofaríngeas , Humanos , Carcinoma Nasofaríngeo/tratamiento farmacológico , Quimioterapia de Inducción , Listas de Espera , Neoplasias Nasofaríngeas/tratamiento farmacológico , Neoplasias Nasofaríngeas/patología , Quimioradioterapia/efectos adversos , Carcinoma/tratamiento farmacológico , Pronóstico , Estudios Retrospectivos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
AIM: Intermedin/adrenomedullin 2 (IMD/ADM2) is a newly discovered peptide closely related to adrenomedullin. We recently reported that IMD/ADM2 gene transfer could significantly reduce renal ischaemia/reperfusion injury. In this study, we evaluated the effect of IMD/ADM2 on cell proliferation and regeneration in a cultured rat renal tubular epithelial cell line (NRK-52E) of hypoxia-reoxygenation (H/R) injury. METHODS: The H/R model in NRK-52E cells consisted of hypoxia for 1 h and reoxygenation for 2 h. IMD/ADM2 was overexpressed in NRK-52E cells using the vector pcDNA3.1-IMD. Enzyme-linked immunosorbent assays were used to measure the concentration of IMD/ADM2 in the culture medium, and real-time PCR and Western blotting were used to determine mRNA and protein levels. In addition, luciferase reporter assays and electrophoretic mobility-shift assays were performed to measure cyclin D1 promoter activity and transcription factor activity. RESULTS: We found that IMD/ADM2 gene transfer markedly promoted cell viability and decreased lactate dehydrogenase (LDH) activity and cell apoptosis compared with that of H/R. IMD/ADM2 increased the phosphorylation of ERK and decreased the phosphorylation of JNK and P38. Furthermore, IMD/ADM2 promoted cell cycle progression with concomitant increases in the levels of cyclin D1 and cyclin E, and these effects were blocked by the inhibition of ERK, or the agonist JNK and P38. IMD/ADM2 also increased cyclin D1 promoter activity and AP-1 DNA-binding activity. CONCLUSIONS: We demonstrated that IMD/ADM2 promotes renal cell proliferation and regeneration after renal H/R injury by upregulating cyclin D1 and that this upregulation seems to be mediated by the ERK, JNK, and P38 MAPK signalling pathways.
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Adrenomedulina/metabolismo , Proliferación Celular , Ciclina D1/metabolismo , Enfermedades Renales/metabolismo , Túbulos Renales/irrigación sanguínea , Túbulos Renales/metabolismo , Regeneración , Daño por Reperfusión/metabolismo , Adrenomedulina/genética , Animales , Apoptosis , Hipoxia de la Célula , Línea Celular , Supervivencia Celular , Ciclina D1/genética , Ciclina E/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Genes Reporteros , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Enfermedades Renales/genética , Enfermedades Renales/patología , Túbulos Renales/patología , L-Lactato Deshidrogenasa/metabolismo , Sistema de Señalización de MAP Quinasas , Fosforilación , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ratas , Daño por Reperfusión/genética , Daño por Reperfusión/patología , Factor de Transcripción AP-1/metabolismo , Transfección , Regulación hacia Arriba , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Antibodies that recognize the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially the neutralizing antibodies, carry great hope in the treatment and final elimination of COVID-19. Driven by a synchronized global effort, thousands of antibodies against the spike protein have been identified during the past two years, with the structural information available at atomistic detail for hundreds of these antibodies. We developed an improved molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) method including explicitly treated interfacial water to calculate the binding free energy between representative antibodies and the receptor binding domain (RBD) domain of SARS-COV-2 spike proteins. We discovered that explicit treatment of water molecules located at the interface between RBD and antibody effectively improves the results for the WT and variants of concern (VOC) systems. Interfacial water molecules, together with surface and internal water molecules, behave drastically from bulk water and exert peculiar impacts on protein dynamics and energy, and thus warrant explicit treatment to complement implicit solvent models. Our results illustrate the importance of including interfacial water molecules to approach efficient and reliable prediction of binding free energy.Communicated by Ramaswamy H. Sarma.
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COVID-19 , Glicoproteína de la Espiga del Coronavirus , Humanos , Agua , SARS-CoV-2 , Anticuerpos Neutralizantes , Unión ProteicaRESUMEN
In this paper, we propose a "reciprocal strategy" that, on the one hand, explores the ability of solid-state nanopores in a homogeneous high-fidelity characterization of nucleic acid assembly and, on the other hand, the formed nucleic acid assembly with a large size serves as an amplifier to provide a highly distinguished and anti-interference signal for molecular sensing. Four-hairpin hybridization chain reaction (HCR) with G-rich tail tags is taken as the proof-of-concept demonstration. G-rich tail tags are commonly used to form G-quadruplex signal probes on the side chain of HCR duplex concatemers. When such G-tailed HCR concatemers translocate the nanopore, abnormal, much higher nanopore signals over normal duplexes can be observed. Combined with atomic force microscopy, we reveal the G-rich tail may easily induce the "intermolecular interaction" between HCR concatemers to form "branched assembly structure (BAS)". To the best of our knowledge, this is the first evidence for the formation BAS of the G tailed HCR concatemers in a homogeneous solution. Systematic nanopore measurements further suggest the formation of these BASs is closely related to the types of salt ions, the amount of G, the concentration of substrate hairpins, the reaction time, and so forth. Under optimized conditions, these BASs can be grown to just the right size without being too large to block the pores, while producing a current 14 times that of conventional double-stranded chains. Here, these very abnormal large current blockages have, in turn, been taken as an anti-interference signal indicator for small targets in order to defend the high noises resulting from co-existing big species (e.g., enzymes or other long double-stranded DNA).
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Técnicas Biosensibles , Nanoporos , Ácidos Nucleicos , ADN/química , Hibridación de Ácido Nucleico/métodos , Técnicas Biosensibles/métodosRESUMEN
As an important enzyme-free amplifier, the hybridization chain reaction (HCR) uses an ssDNA to trigger cycled displacement interactions between substrate hairpins and finally form elongated dsDNA concatamer mixtures. In many cases, to provide a signal probe or advanced function, additional oligonucleotides (named hairpin tails) have to be extended upon classic HCR hairpin substrates, but by doing so the HCR assembly efficiency and signal-to-noise ratio (SNR) may get seriously reduced. In this Article, a rational and general model that may guide the study on HCR functionalization and signaling efficiency is provided. We rationally design a four-hairpin model HCR system (4H-HCR) in which one or more hairpin substrates are appended with additional tails as a signaling probe. After HCR assembly, two adjacent tails are supposedly integrating into a full G-quadruplex structure to provide the evidence or signal for the assembly. A systematic study has been applied to reveal the relationship between the "tail-design" with assembly efficiency and SNR. A clear design rule-set guiding the optimized assembly and signal has been provided for traditional electrophoresis and G-quadruplex-enhanced fluorescence signal. Importantly, solid-state nanopore single molecular detection has been innovatively introduced and recommended as an "antirisk" and "mutual benefit" readout to traditional G-quadruplex signaling. Nanopore detection can provide a clear signal distinguished before and after the HCR reaction, especially when the traditional G-quadruplex-enhanced signal only provides low SNR. The G-quadruplex, in turn, may enhance the nanopore signal amplitude via increasing the diameter of the HCR products.
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ADN/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico , ADN de Cadena Simple/metabolismo , Colorantes Fluorescentes/química , G-Cuádruplex , Nanoporos , Protoporfirinas/química , Relación Señal-Ruido , Espectrometría de FluorescenciaRESUMEN
Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.
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Sustancias Macromoleculares/análisis , Nanoporos , Aptámeros de Nucleótidos/análisis , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/metabolismo , Cocaína/metabolismo , ADN Concatenado/análisis , ADN Concatenado/genética , ADN Concatenado/metabolismo , Electroforesis , Formamidas/química , Sustancias Macromoleculares/química , Hibridación de Ácido Nucleico/efectos de los fármacosRESUMEN
Recent advances have proven that using solid-state nanopores is a promising single molecular technique to enrich the DNA assembly signaling library. Other than using them for distinguishing structures, here we innovatively adapt solid-state nanopores for use in analyzing assembly mixtures, which is usually a tougher task for either traditional characterization techniques or nanopores themselves. A trigger induced DNA step polymerization (SP-CHA), producing three-way-DNA concatemers, is designed as a model. Through counting and integrating the translocation-induced current block when each concatemer passes through a glass conical glass nanopore, we propose an electrophoresis-gel like, but homogeneous, quantitative method that can comprehensively profile the "base-pair distribution" of SP-CHA concatemer mixtures. Due to the higher sensitivity, a number of super long concatemers that were previously difficult to detect via gel electrophoresis are also revealed. These ultra-concatemers, longer than 2 kbp, could provide a much enhanced signal-to-noise ratio for nanopores and are thus believed to be more accurate indicators for the existence of a trigger, which may be of benefit for further applications, such as molecular machines or biosensors.
RESUMEN
Glass capillary-based nanopore is exploited for single-molecule conformational sensing of multiarm DNA concatemers during translocation. Both translocation frequency and orientation preference were found related with the number of arms of the DNA concatemers.
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ADN Concatenado/análisis , Técnicas Electroquímicas/métodos , Vidrio/química , Nanoporos , Conformación de Ácido NucleicoRESUMEN
Recent advances have proven solid-state nanopores as a powerful analysis platform that enables label-free and separation-free single-molecule analysis. However, the relatively low resolution still limits its application because many chemicals or targets with small sizes could not be recognized in a label-free condition. In this paper, we provide a possible solution that uses solid-state nanopores for small species signaling via the transition of huge DNA assembly products. DNAzyme responding to metal ions and the hybridization chain reaction (HCR) generating nanopore-detectable dsDNA concatamers are used as the transition model set. By the two-step DNAzyme-HCR transition, Pb2+ that was too tiny to be sensed was successfully recognized by the nanopore. The whole process happened in a completely homogeneous solution without any chemical modification. During condition optimization, we also discussed one possible application challenge that may affect the HCR signal-background distinction. Solid-state nanopores provide a potential solution to this challenge due to its ability to profile product length or even 3D structure information.
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ADN Catalítico/química , Plomo/análisis , Metales , Nanoporos , Ribonucleasas/química , Transducción de Señal , Hibridación de Ácido NucleicoRESUMEN
Highly pathogenic avian influenza A virus H5N1 continues to spread among poultry and has frequently broken the species barrier to humans. Recent studies have shown that a laboratory-mutated or reassortant H5N1 virus bearing hemagglutinin (HA) with as few as four or five mutations was capable of transmitting more efficiently via respiratory droplets between ferrets, posing a serious threat to public health and underscoring the priority of effective vaccines and therapeutics. In this study, we identified a novel monoclonal antibody (mAb) named HAb21, that has a broadly neutralizing activity against all tested strains of H5N1 covering clades 0, 1, 2.2, 2.3.4, and 2.3.2.1. Importantly, HAb21 efficiently neutralized diverse H5N1 variants with single or combination forms of mutations capable of airborne transmission. We demonstrated that HAb21 blocked viral entry during the receptor-binding step by targeting a previously uncharacterized epitope at the tip of the HA head. This novel epitope closely neighbors the receptor-binding site (RBS) and the interface of HA trimer and is highly conserved among divergent H5N1 strains. Our studies provide a new tool for use either for therapeutic purposes or as a basis of vaccine development.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antivirales/farmacología , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N1 del Virus de la Influenza A/fisiología , Internalización del Virus/efectos de los fármacos , Animales , Línea Celular , Modelos Animales de Enfermedad , Perros , Hurones , Humanos , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Gripe Humana , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virologíaRESUMEN
The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
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Bacteriófagos/genética , Glicoproteínas de Membrana/genética , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Proteínas del Envoltorio Viral/genética , Anticuerpos Antivirales/inmunología , Bacteriófagos/metabolismo , Epítopos/genética , Epítopos/inmunología , Humanos , Glicoproteínas de Membrana/inmunología , Biblioteca de Péptidos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Chromite ore processing residues (COPR) is the source of the Cr(VI) contamination in the environment. Pannonibacter phragmitetus BB was used to treat two different types of COPRs in this research. The water-soluble Cr(VI) of COPR A and B is 3,982.9 and 1,181.4 mg/kg, respectively. In the column biotreatment process, P. phragmitetus BB can reduce Cr(VI) in the leachate to an undetectable level at the flow rate of 1 and 2 ml/min. In the direct biotreatment process, Cr(VI) in the liquid supernatant of COPR A and B decreased from 265 and 200 mg/l to 145 and 40 mg/kg after 240 h of incubation. In one-step and two-step biotreatment processes, Cr(VI) in the liquid supernatant of both COPRs can be reduced to an undetectable level. The toxicity characteristic leaching procedure results indicate that the Cr(VI) concentration of treated COPR A (3.48 mg/l) is lower than the identification standards for hazardous wastes of China (5 mg/l) (GB 5085.6-2007). The information obtained in this study has significance for the application of P. phragmitetus BB to remediate COPR contamination.
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Compuestos de Cromo/metabolismo , Rhodobacteraceae/metabolismo , Compuestos de Cromo/toxicidad , Residuos Industriales/análisis , Minería , Administración de Residuos/métodosRESUMEN
BACKGROUND: It is necessary to develop some innovative methods to reveal and discover the novel (SLE)-related protein molecules. In the present study, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) was employed to detect the differential expression of serum polypeptides in the patients with systemic lupus erythematosus (SLE) presenting with a family history or complicating with kidney injury so as to identify the proteins associated with the genetic factors and kidney injury in SLE. METHODS: The subjects recruited were divided into four groups, that is, a group of SLE patients with both family history and kidney injury, a group of SLE patients with only kidney injury but no family history, a group of SLE patients with neither family history nor kidney injury, and a control group consisting of healthy volunteers. By adopting MALDI-TOF MS analysis, the serum samples obtained from the three groups of SLE patients were examined and compared with those from the control group; the categorized peptide fingerprint profile was established via the biological data collected from the samples. RESULTS: The expression of protein with a m/z of 4207 Da increased significantly in SLE patients; the protein with a m/z of 2658 Da was expressed in all SLE patients; three proteins (with m/z of 1465, 5332, and 5900 Da respectively) were expressed in the SLE patients complicated with kidney injury and the protein with a m/z of 1943 Da was expressed in SLE patients with family history. CONCLUSION: A number of differential proteins were successfully detected and identified through MALDI-TOF MS detection and these proteins may be associated with the genetic basis of SLE and the complicating kidney injury.