Comprehensive Study on Aptamers and Aptamer-based Assays

Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.

Comprehensive Study on Aptamers and Aptamer-based Assays

Antibodies are the gold standard molecular recognition elements and a cornerstone of molecular biology. They are used in immunoassays to precisely measure a specific analyte, but certain targets are especially challenging. Difficult targets include small molecules and molecules that do not induce an immune response. Aptamers are short oligonucleotides that can form 3-dimensional structures and bind targets with high specificity. Aptamers are smaller and more flexible than antibodies and could therefore solve this problem. In contrast to antibodies, aptamers are synthetically produced, so they can have affinity for molecules that do not induce an immune response. This also makes them cheaper, faster and more ethical to produce. They are also easily modified and have the ability to renature and can therefore be reused.  Our conclusions are that aptamers can outperform antibodies, especially for small molecule targets, and that the synthetic production of aptamers gives them a further advantage over antibodies. Our report compares several different types of detection methods that use aptamers and we conclude that fluorescence-based methods are the most easy to use with basic lab equipment, can be made similar to the ELISA kits in addition to giving highly sensitive detection. We describe a variety of fluorescence-based detection strategies but the optimal method will depend on the specific aptamer and target. The report also includes an ethical analysis where antibodies and aptamers are compared. This report is commissioned by Mercodia AB, a company that develops, manufactures and distributes immunoassays for biomarkers within the field of metabolic disorders. They commissioned this report in order to give an overview of how aptamers interact with their target, and also to compare aptamer-based detection strategies with sensitivity prioritized over selectivity.  This was done by literature research.

Validation of Boundary-Layer-Transition Computations for a Rotor with Axial Inflow

Boundary-layer-transition computations are performed using the in-house finite-volume solvers elsA by ONERA and TAU by DLR. Reynolds-averaged Navier-Stokes simulations, using the Langtry-Menter model as well as semiempirical transition criteria, are presented using both solvers for a rotor in climb. The numerical results are compared to temperature-sensitive paint experiments conducted at DLR's rotor test facility. Concerning the Langtry-Menter computation, transition occurs with both solvers due to laminar separation close to the trailing edge, further downstream than seen in the experiment. Semi-empirical transition criteria predict transition within both codes due to laminar separation, which was not detected in the experiments. When only considering the AHD criterion, the transition locations within the numerical simulations and the experiment are in good agreement along the entire span in all three considered test cases. In addition, numerical results are presented for a test case with cyclic pitch. These unsteady boundary-layer transition computations are carried out using the semi-empirical transition criteria approach of DLR-TAU. In accordance with the static test cases, the results are promising, as long as the laminar-separation criterion is deactivated

Assessment of Boundary Layer Transition Prediction Methods for Rotating Blades

Boundary-layer-transition computations are performed using the in-house finite-volume solvers elsA by ONERA and TAU by DLR. Reynolds-averaged Navier-Stokes simulations, using the Langtry-Menter model as well as semiempirical transition criteria, are presented using both solvers for a rotor in climb. The numerical results are compared to temperature-sensitive paint experiments conducted at DLR's rotor test facility. Concerning the Langtry-Menter computation, transition occurs with both solvers due to laminar separation close to the trailing edge, further downstream than seen in the experiment. The transition locations using AHD semi-empirical transition criterion are in good agreement along the entire span in all three considered test cases. In addition, numerical results are presented for a test case with cyclic pitch. These unsteady boundary-layer transition computations are carried out using the semi-empirical transition criteria approach of DLR-TAU. In accordance with the static test cases, the results are promising, as long as the laminar-separation criterion is deactivated

Characterization of New Monoclonal PF4-Specific Antibodies as Useful Tools for Studies on Typical and Autoimmune Heparin-Induced Thrombocytopenia

International audienceBackground Heparin-induced thrombocytopenia (HIT) is typically caused by plateletactivating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin (H). Much less frequent "autoimmune" HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs. Objectives To characterize 1E12, 1C12, and 2E1 in comparison to the heparindependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3). Methods Interactions of Abs with PF4 and PF4/H were studied by enzyme-linkedimmunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method). Results 1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin. Conclusion 1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs

Exploratory Data Analysis of Cell and Mitochondrial High-Fat, High-Sugar Toxicity on Human HepG2 Cells

Non-alcoholic steatohepatitis (NASH), one of the deleterious stages of non-alcoholic fatty liver disease, remains a significant cause of liver-related morbidity and mortality worldwide. In the current work, we used an exploratory data analysis to investigate time-dependent cellular and mitochondrial effects of different supra-physiological fatty acids (FA) overload strategies, in the presence or absence of fructose (F), on human hepatoma-derived HepG2 cells. We measured intracellular neutral lipid content and reactive oxygen species (ROS) levels, mitochondrial respiration and morphology, and caspases activity and cell death. FA-treatments induced a time-dependent increase in neutral lipid content, which was paralleled by an increase in ROS. Fructose, by itself, did not increase intracellular lipid content nor aggravated the effects of palmitic acid (PA) or free fatty acids mixture (FFA), although it led to an up-expression of hepatic fructokinase. Instead, F decreased mitochondrial phospholipid content, as well as OXPHOS subunits levels. Increased lipid accumulation and ROS in FA-treatments preceded mitochondrial dysfunction, comprising altered mitochondrial membrane potential (ΔΨm) and morphology, and decreased oxygen consumption rates, especially with PA. Consequently, supra-physiological PA alone or combined with F prompted the activation of caspase pathways leading to a time-dependent decrease in cell viability. Exploratory data analysis methods support this conclusion by clearly identifying the effects of FA treatments. In fact, unsupervised learning algorithms created homogeneous and cohesive clusters, with a clear separation between PA and FFA treated samples to identify a minimal subset of critical mitochondrial markers in order to attain a feasible model to predict cell death in NAFLD or for high throughput screening of possible therapeutic agents, with particular focus in measuring mitochondrial function

Acute Toxicity Grade 3 and 4 After Irradiation in Children and Adolescents: Results From the IPPARCA Collaboration

Purpose: In the context of oncologic therapy for children, radiation therapy is frequently indicated. This study identified the frequency of and reasons for the development of high-grade acute toxicity and possible sequelae. Materials and Methods: Irradiated children have been prospectively documented since 2001 in the Registry for the Evaluation of Side Effects After Radiation in Childhood and Adolescence (RiSK) database in Germany and since 2008 in the registry for radiation therapy toxicity (RADTOX) in Sweden. Data were collected using standardized, published forms. Toxicity classification was based on Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer criteria. Results: As of June 2013, 1500 children have been recruited into the RiSK database and 485 into the RADTOX registry leading to an analysis population of 1359 patients (age range 0-18). A total of 18.9% (n=257) of all investigated patients developed high-grade acute toxicity (grades 3/4). High-grade toxicity of the bone marrow was documented for 63.8% (n=201) of those patients, oral mucositis for 7.6% (n=24), and dermatitis for 7.6% (n=24). Patients with high-grade acute toxicity received concomitant chemotherapy more frequently (56%) than patients with no or lower acute toxicity (31.5%). In multivariate analyses, concomitant chemotherapy, diagnosis of Ewing sarcoma, and total radiation dose showed a statistically noticeable effect (P <=.05) on acute toxicity, whereas age, concomitant chemotherapy, Hodgkin lymphoma, Ewing sarcoma, total radiation dose, and acute toxicity influenced the time until maximal late toxicity. Conclusions: Generally, high-grade acute toxicity after irradiation in children and adolescence occurs in a moderate proportion of patients (18.9%). As anticipated, the probability of acute toxicity appeared to depend on the prescribed dose as well as concomitant chemotherapy. The occurrence of chronic toxicity correlates with the prior acute toxicity grade. Age seems to influence the time until maximal late toxicity but not the development of acute toxicity. (c) 2016 Elsevier Inc. All rights reserved

Radiation synergizes with antitumor activity of CD13-targeted tissue factor in a HT1080 xenograft model of human soft tissue sarcoma.

BACKGROUND:Truncated tissue factor (tTF) retargeted by NGR-peptides to aminopeptidase N (CD13) in tumor vasculature is effective in experimental tumor therapy. tTF-NGR induces tumor growth inhibition in a variety of human tumor xenografts of different histology. To improve on the therapeutic efficacy we have combined tTF-NGR with radiotherapy. METHODS:Serum-stimulated human umbilical vein endothelial cells (HUVEC) and human HT1080 sarcoma cells were irradiated in vitro, and upregulated early-apoptotic phosphatidylserine (PS) on the cell surface was measured by standard flow cytometry. Increase of cellular procoagulant function in relation to irradiation and PS cell surface concentration was measured in a tTF-NGR-dependent Factor X activation assay. In vivo experiments with CD-1 athymic mice bearing human HT1080 sarcoma xenotransplants were performed to test the systemic therapeutic effects of tTF-NGR on tumor growth alone or in combination with regional tumor ionizing radiotherapy. RESULTS:As shown by flow cytometry with HUVEC and HT1080 sarcoma cells in vitro, irradiation with 4 and 6 Gy in the process of apoptosis induced upregulation of PS presence on the outer surface of both cell types. Proapoptotic HUVEC and HT1080 cells both showed significantly higher procoagulant efficacy on the basis of equimolar concentrations of tTF-NGR as measured by FX activation. This effect can be reverted by masking of PS with Annexin V. HT1080 human sarcoma xenografted tumors showed shrinkage induced by combined regional radiotherapy and systemic tTF-NGR as compared to growth inhibition achieved by either of the treatment modalities alone. CONCLUSIONS:Irradiation renders tumor and tumor vascular cells procoagulant by PS upregulation on their outer surface and radiotherapy can significantly improve the therapeutic antitumor efficacy of tTF-NGR in the xenograft model used. This synergistic effect will influence design of future clinical combination studies

Antidepressant drugs act by directly binding to TRKB neurotrophin receptors

It is unclear how binding of antidepressant drugs to their targets gives rise to the clinical antidepressant effect. We discovered that the transmembrane domain of tyrosine kinase receptor 2 (TRKB), the brain-derived neurotrophic factor (BDNF) receptor that promotes neuronal plasticity and antidepressant responses, has a cholesterol-sensing function that mediates synaptic effects of cholesterol. We then found that both typical and fast-acting antidepressants directly bind to TRKB, thereby facilitating synaptic localization of TRKB and its activation by BDNF. Extensive computational approaches including atomistic molecular dynamics simulations revealed a binding site at the transmembrane region of TRKB dimers. Mutation of the TRKB antidepressant-binding motif impaired cellular, behavioral, and plasticity-promoting responses to antidepressants in vitro and in vivo. We suggest that binding to TRKB and allosteric facilitation of BDNF signaling is the common mechanism for antidepressant action, which may explain why typical antidepressants act slowly and how molecular effects of antidepressants are translated into clinical mood recovery.Peer reviewe