scholarly journals Real-Time PCR-Coupled CE-SELEX for DNA Aptamer Selection

2012 ◽  
Vol 2012 ◽  
pp. 1-9 ◽  
Author(s):  
Patrick Ruff ◽  
Rekha B. Pai ◽  
Francesca Storici
Keyword(s):  
Real Time ◽  
High Specificity ◽  
Cost Effective ◽  
Dna Aptamer ◽  
Initial Time ◽  
Mobility Shift ◽  
Traditional Procedure ◽  
Supershift Assay ◽  
Exponential Enrichment ◽  
Electrophoretic Mobility Shift Assays

Aptamers are short nucleic acid or peptide sequences capable of binding to a target molecule with high specificity and affinity. Also known as “artificial antibodies,” aptamers provide many advantages over antibodies. One of the major hurdles to aptamer isolation is the initial time and effort needed for selection. The systematic evolution of ligands by exponential enrichment (SELEX) is the traditional procedure for generating aptamers, but this process is lengthy and requires a large quantity of target and starting aptamer library. A relatively new procedure for generating aptamers using capillary electrophoresis (CE), known as CE-SELEX, is faster and more efficient than SELEX but requires laser-induced fluorescence (LIF) to detect the aptamer-target complexes. Here, we implemented an alternative system without LIF using real-time- (RT-) PCR to indirectly measure aptamer-target complexes. In three rounds of selection, as opposed to ten or more rounds common in SELEX protocols, a specific aptamer for bovine serum albumin (BSA) was obtained. The specificity of the aptamer to BSA was confirmed by electrophoretic mobility shift assays (EMSAs), an unlabeled competitor assay, and by a supershift assay. The system used here provides a cost effective and a highly efficient means of generating aptamers.

scholarly journals The Sequence-specific DNA Binding of NF-κB Is Reversibly Regulated by the Automodification Reaction of Poly (ADP-ribose) Polymerase 1

2001 ◽  
Vol 276 (50) ◽  
pp. 47664-47670 ◽  
Author(s):  
Woo-Jin Chang ◽  
Rafael Alvarez-Gonzalez
Keyword(s):  
Dna Binding ◽  
Hela Cells ◽  
Specific Binding ◽  
High Specificity ◽  
Immunoblot Analysis ◽  
Mobility Shift ◽  
Electrophoretic Mobility Shift Assays ◽  
Parp 1

Recent studies suggest that the synthesis of protein-bound ADP-ribose polymers catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1) regulates eucaryotic gene expression, including the NF-κB-dependent pathway. Here, we report the molecular mechanism by which PARP-1 activates the sequence-specific binding of NF-κB to its oligodeoxynucleotide. We co-incubated pure recombinant human PARP-1 and the p50 subunit of NF-κB (NF-κB-p50) in the presence or absence of βNAD+in vitro.Electrophoretic mobility shift assays showed that, when PARP-1 was present, NF-κB-p50 DNA binding was dependent on the presence of βNAD+. DNA binding by NF-κB-p50 was not efficient in the absence of βNAD+. In fact, the binding was not efficient in the presence of 3-aminobenzamide (3-AB) either. Thus, we conclude that NF-κB-p50 DNA binding is protein-poly(ADP-ribosyl)ation dependent. Co-immunoprecipitation and immunoblot analysis revealed that PARP-1 physically interacts with NF-κB-p50 with high specificity in the absence of βNAD+. Because NF-kB-p50 was not an efficient covalent target for poly(ADP-ribosyl)ation, our results are consistent with the conclusion that the auto-poly(ADP-ribosyl)ation reaction catalyzed by PARP-1 facilitates the binding of NF-κB-p50 to its DNA by inhibiting the specific protein·protein interactions between NF-κB-p50 and PARP-1. We also report the activation of NF-κB DNA binding by the automodification reaction of PARP-1 in cultured HeLa cells following exposure to H2O2. In these experiments, preincubation of HeLa cells with 3-AB, prior to oxidative damage, strongly inhibited NF-κB activationin vivoas well.

scholarly journals Unraveling Determinants of Affinity Enhancement in Dimeric Aptamers for a Dimeric Protein

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Sepehr Manochehry ◽  
Erin M. McConnell ◽  
Yingfu Li
Keyword(s):  
Binding Site ◽  
De Novo ◽  
Mobility Shift ◽  
High Affinity ◽  
Two Component ◽  
Systematic Evolution ◽  
Dimeric Protein ◽  
Enrichment Experiments ◽  
Exponential Enrichment ◽  
Electrophoretic Mobility Shift Assays

AbstractHigh-affinity aptamers can be derived de novo by using stringent conditions in SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments or can be engineered post SELEX via dimerization of selected aptamers. Using electrophoretic mobility shift assays, we studied a series of heterodimeric and homodimeric aptamers, constructed from two DNA aptamers with distinct primary sequences and secondary structures, previously isolated for VEGF-165, a homodimeric protein. We investigated four factors envisaged to impact the affinity of a dimeric aptamer to a dimeric protein: (1) length of the linker between two aptamer domains, (2) linking orientation, (3) binding-site compatibility of two component aptamers in a heterodimeric aptamer, and (4) steric acceptability of the two identical aptamers in a homodimeric aptamer. All heterodimeric aptamers for VEGF-165 were found to exhibit monomeric aptamer-like affinity and the lack of affinity enhancement was attributed to binding-site overlap by the constituent aptamers. The best homodimeric aptamer showed 2.8-fold better affinity than its monomeric unit (Kd = 13.6 ± 2.7 nM compared to 37.9 ± 14 nM), however the barrier to further affinity enhancement was ascribed to steric interference of the constituent aptamers. Our findings point to the need to consider the issues of binding-site compatibility and spatial requirement of aptamers for the development of dimeric aptamers capable of bivalent recognition. Thus, determinants highlighted herein should be assessed in future multimerization efforts.

scholarly journals Cost-Effective Pooling of DNA from Nasopharyngeal Swab Samples for Large-Scale Detection of Bacteria by Real-Time PCR

2014 ◽  
Vol 53 (3) ◽  
pp. 1002-1004 ◽  
Author(s):  
Sophie Edouard ◽  
Elsa Prudent ◽  
Philippe Gautret ◽  
Ziad A. Memish ◽  
Didier Raoult
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Large Scale ◽  
High Sensitivity ◽  
High Specificity ◽  
Cost Effective ◽  
Nasopharyngeal Swab ◽  
Rt Pcr ◽  
Scale Detection ◽  
The Cost

We investigated the potential of pooling DNA from nasopharyngeal specimens to reduce the cost of real-time PCR (RT-PCR) for bacterial detection. Lyophilization is required to reconcentrate DNA. This strategy yields a high specificity (86%) and a high sensitivity (96%). We estimate that compared to individual testing, 37% fewer RT-PCR tests are needed.

531: Quantitative Real-Time RT-PCR for AMACR Distinguishes Prostate Cancer with High Specificity From Benign Lesions

2005 ◽  
Vol 173 (4S) ◽  
pp. 145-145 ◽  
Author(s):  
Martin Schostak ◽  
Hans Krause ◽  
Jens Köllermann ◽  
Mark Schrader ◽  
Bernd Straub ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Real Time ◽  
High Specificity ◽  
Rt Pcr ◽  
Benign Lesions

Antiparasitic Treatment of Patients with P. falciparum Malaria Reduces the Ability of Patient Serum to Induce Tissue Factor by Decreasing NF-κB Activation

1995 ◽  
Vol 73 (01) ◽  
pp. 039-048 ◽  
Author(s):  
A Bierhaus ◽  
Ch J Hemmer ◽  
N Mackman ◽  
R Kutob ◽  
R Ziegler ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Falciparum Malaria ◽  
De Novo ◽  
Neutralizing Antibody ◽  
Mobility Shift ◽  
Before And After ◽  
Tf Gene ◽  
Electrophoretic Mobility Shift Assays ◽  
Antiparasitic Treatment ◽  
Stimulation Of

SummarySerum from patients with P. falciparum malaria at day 1 (pretherapy) induces tissue factor (TF) in cultured endothelial cells. TF induction depends on de novo transcription as shown in Nuclear Run On assays. Electrophoretic mobility shift assays demonstrated binding of AP-1 and NF- κB/Rel proteins to their recognition sites in the TF promotor. After therapy (day 28), stimulation of TF antigen by patient serum is reduced by 70%. When serum obtained before and after therapy was compared, a decrease of NF-κB activation was evident. Activation of NF-κB-like proteins was in part dependent on TNFα in patient serum, since a TNFα neutralizing antibody reduced induction of TF transcription and translation and induction of NF-κB-like proteins. Induction of TF activity was suppressed by pDTC, an inhibitor of NF-κB activation. When different promotor constructs of the TF gene were tested, induction was dependent upon the presence of the intact NF-κB-like binding site in the TF promotor. A mutant with deleted NF-κB, but intact AP-1 sites was not inducible. Mutation of the AP-1 sites did not prevent induction, but reduced inducibility by pretherapy serum. Therefore, NF-κB/Rel proteins are responsible for induction of TF transcription by pretherapy serum, but AP-1 is needed for highest inducibility. The effect of antiparasitic therapy on the induction of TF by serum from patients with complicated P. falciparum malaria is dependent on a therapy-mediated loss of activation of NF-κB-like proteins in post-treatment patient serum.

scholarly journals Modeling Fused Filament Fabrication using Artificial Neural Networks

2021 ◽  
Author(s):  
Paul Oehlmann ◽  
Paul Osswald ◽  
Juan Camilo Blanco ◽  
Martin Friedrich ◽  
Dominik Rietzel ◽  
...  
Keyword(s):  
Real Time ◽  
Large Scale ◽  
Cost Effective ◽  
Print Quality ◽  
Ann Model ◽  
Production Environment ◽  
Fused Filament Fabrication ◽  
Artificial Neural ◽  
Using Data ◽  
Artificial Neural Network Ann

AbstractWith industries pushing towards digitalized production, adaption to expectations and increasing requirements for modern applications, has brought additive manufacturing (AM) to the forefront of Industry 4.0. In fact, AM is a main accelerator for digital production with its possibilities in structural design, such as topology optimization, production flexibility, customization, product development, to name a few. Fused Filament Fabrication (FFF) is a widespread and practical tool for rapid prototyping that also demonstrates the importance of AM technologies through its accessibility to the general public by creating cost effective desktop solutions. An increasing integration of systems in an intelligent production environment also enables the generation of large-scale data to be used for process monitoring and process control. Deep learning as a form of artificial intelligence (AI) and more specifically, a method of machine learning (ML) is ideal for handling big data. This study uses a trained artificial neural network (ANN) model as a digital shadow to predict the force within the nozzle of an FFF printer using filament speed and nozzle temperatures as input data. After the ANN model was tested using data from a theoretical model it was implemented to predict the behavior using real-time printer data. For this purpose, an FFF printer was equipped with sensors that collect real time printer data during the printing process. The ANN model reflected the kinematics of melting and flow predicted by models currently available for various speeds of printing. The model allows for a deeper understanding of the influencing process parameters which ultimately results in the determination of the optimum combination of process speed and print quality.

scholarly journals An Innovative and Cost-Advantage CAD Solution for Cubitus Varus Surgical Planning in Children

2021 ◽  
Vol 11 (9) ◽  
pp. 4057
Author(s):  
Leonardo Frizziero ◽  
Gian Maria Santi ◽  
Christian Leon-Cardenas ◽  
Giampiero Donnici ◽  
Alfredo Liverani ◽  
...  
Keyword(s):  
Computer Aided Design ◽  
Low Cost ◽  
Cost Effective ◽  
Varus Deformity ◽  
Patient Specific ◽  
Cubitus Varus ◽  
Surgical Guide ◽  
Traditional Procedure ◽  
Custom Made ◽  
Patient Specific Instruments

The study of CAD (computer aided design) modeling, design and manufacturing techniques has undergone a rapid growth over the past decades. In medicine, this development mainly concerned the dental and maxillofacial sectors. Significant progress has also been made in orthopedics with pre-operative CAD simulations, printing of bone models and production of patient-specific instruments. However, the traditional procedure that formulates the surgical plan based exclusively on two-dimensional images and interventions performed without the aid of specific instruments for the patient and is currently the most used surgical technique. The production of custom-made tools for the patient, in fact, is often expensive and its use is limited to a few hospitals. The purpose of this study is to show an innovative and cost-effective procedure aimed at prototyping a custom-made surgical guide for address the cubitus varus deformity on a pediatric patient. The cutting guides were obtained through an additive manufacturing process that starts from the 3D digital model of the patient’s bone and allows to design specific models using Creo Parametric. The result is a tool that adheres perfectly to the patient’s bone and guides the surgeon during the osteotomy procedure. The low cost of the methodology described makes it worth noticing by any health institution.

scholarly journals Cost-Effective Real-Time Metabolic Profiling of Cancer Cell Lines for Plate-Based Assays

Chemosensors ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 139
Author(s):  
Wiktoria Blaszczak ◽  
Zhengchu Tan ◽  
Pawel Swietach
Keyword(s):  
Real Time ◽  
Cell Lines ◽  
Acid Production ◽  
Metabolic Regulation ◽  
Cost Effective ◽  
Culture Conditions ◽  
Metabolic Inhibitors ◽  
Metabolic Phenotype ◽  
Ductal Adenocarcinoma ◽  
Oxygen Utilization

A fundamental phenotype of cancer cells is their metabolic profile, which is routinely described in terms of glycolytic and respiratory rates. Various devices and protocols have been designed to quantify glycolysis and respiration from the rates of acid production and oxygen utilization, respectively, but many of these approaches have limitations, including concerns about their cost-ineffectiveness, inadequate normalization procedures, or short probing time-frames. As a result, many methods for measuring metabolism are incompatible with cell culture conditions, particularly in the context of high-throughput applications. Here, we present a simple plate-based approach for real-time measurements of acid production and oxygen depletion under typical culture conditions that enable metabolic monitoring for extended periods of time. Using this approach, it is possible to calculate metabolic fluxes and, uniquely, describe the system at steady-state. By controlling the conditions with respect to pH buffering, O2 diffusion, medium volume, and cell numbers, our workflow can accurately describe the metabolic phenotype of cells in terms of molar fluxes. This direct measure of glycolysis and respiration is conducive for between-runs and even between-laboratory comparisons. To illustrate the utility of this approach, we characterize the phenotype of pancreatic ductal adenocarcinoma cell lines and measure their response to a switch of metabolic substrate and the presence of metabolic inhibitors. In summary, the method can deliver a robust appraisal of metabolism in cell lines, with applications in drug screening and in quantitative studies of metabolic regulation.

scholarly journals Traceable Air Baggage Handling System Based on RFID Tags in the Airport

2008 ◽  
Vol 3 (1) ◽  
pp. 106-115 ◽  
Author(s):  
Ting Zhang ◽  
Yuanxin Ouyang ◽  
Yang He
Keyword(s):  
Supply Chain ◽  
Real Time ◽  
Recognition Rate ◽  
Cost Effective ◽  
Object Identification ◽  
Rfid Tags ◽  
Handling System ◽  
International Airport ◽  
Management Efficiency ◽  
Baggage Handling

The RFID is not only a feasible, novel, and cost-effective candidate for daily object identification but it is also considered as a significant tool to provide traceable visibility along different stages of the aviation supply chain. In the air baggage handing application, the RFID tags are used to enhance the ability for baggage tracking, dispatching and conveyance so as to improve the management efficiency and the users’ satisfaction. We surveyed current related work and introduce the IATA RP1740c protocol used for the standard to recognize the baggage tags. One distributed aviation baggage traceable application is designed based on the RFID networks. We describe the RFID-based baggage tracking experiment in the BCIA (Beijing Capital International Airport). In this experiment the tags are sealed in the printed baggage label and the RFID readers are fixed in the certain interested positions of the BHS in the Terminal 2. We measure the accurate recognition rate and monitor the baggage’s real-time situation on the monitor’s screen. Through the analysis of the measured results within two months we emphasize the advantage of the adoption of RFID tags in this high noisy BHS environment. The economical benefits achieved by the extensive deployment of RFID in the baggage handing system are also outlined.

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