Electrochemical enzyme-linked immunosorbent assay (e-ELISA) for parasitic nematode: Ostertagia ostertagi (brown stomach worm) infections in dairy cattle

A sensitive electrochemical immunoassay (e-ELISA) has been developed for the detection of the gastrointestinal parasitic nematode Ostertagia ostertagi (brown stomach worm) in infected and control serum samples. An antigen-indirect immunoassay format was employed to detect the presence of O. ostertagi antibodies, coupled with an anti-species monoclonal horseradish peroxidase (HRP) conjugate. ABTS (2,2\u27-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) and TMB (3,3\u27,5,5\u27-tetramethylbenzidine/hydrogen peroxide) were investigated as both chromogenic visualising reagents for optical ELISA and electroactive substrates for electrochemical ELISA in the HRP catalysed oxidation reaction. Coulometry was applied for the detection of O. ostertagi antibodies (via TMB electrochemistry) and compared with the commercial optical ELISA (ABTS based SVANOVIR® O. ostertagi-Ab ELISA kit). Cost-effective in-house sensors were designed and fabricated using polyester and chemical adhesive materials with the aid of stencil printing and laser machining techniques. The performance of the electrochemical ELISA and sensor was evaluated by investigating redox mediators (ABTS vs. TMB), stop solutions (sodium dodecyl sulfate vs. sulfuric acid) and incubation times (150 min vs. 70 min vs. 25 min). For a total assay incubation time of 70 minutes, the TMB/H2SO4 based e-ELISA was able to differentiate between positive (P) and negative (N) control serum samples, with a P/N70 control ratio 1.6 times higher than that of optical ELISA (TMB/H2SO4 combination) and 2.9 times higher than that of the commercial ELISA kit (ABTS/SDS combination). Furthermore, the e-ELISA approach is quicker and required only 25 min (total incubation time) with even better response (P/N25 = 14.7), which is approximately 4-fold higher than the optical immunoassay (P/N25 = 3.8). The proposed e-ELISA is specific (selective Ab-Ag interactions) and highly sensitive - capable of detecting up to 16-fold dilutions of a positive control serum sample. The electrochemical ELISA approach has the potential for rapid sample screening in a portable, disposable format, contributing to the quest for effective prevention and control of parasitic Ostertagia ostertagi infections in cattle

Comparative Study of the Electrochemical Signal of Neonicotinoids and Tetronic Acid Amides on Screen Printed Electrodes With and Without the Use Of N2a Cells.

The extensive use of pesticides in agriculture has caused significant concern in public health therefore cell-based sensors have been proved as potentially useful method for studying their effects. The objective of this work was to investigate the possibility of using carbon screen printed electrodes (SPE) in combination with the use of N2a cells for the direct voltammetric determination of 5 neonicotinoids (imidacloprid, clothianidin, thiacloprid, acetamiprid and thiamethoxam) and 3 tetronic acid amide insecticides (spiromesifen, spirodiclofen and spirotetramat). The insecticide cytotoxicity in N2a cells was determined after 30 min treatment with concentrations 3, 10, 30 and 100 μM by the propidium iodide (PI) uptake assay. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were performed to compare signals from plain carbon screen printed electrodes and from N2a cells

Spheroid-3D and Monolayer-2D Intestinal Electrochemical Biosensor for Toxicity/Viability Testing: Applications in Drug Screening, Food Safety, and Environmental Pollutant Analysis

The rise of three-dimensional cell culture systems that provide in vivo-like environments for pharmaco-toxicological models has prompted the need for simple and robust viability assays suitable for complex cell architectural structures. This study addresses that challenge with the development of an in vitro enzyme based electrochemical sensor for viability/cytotoxicity assessment of two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture formats. The biosensor measures the cell viability/toxicity via electrochemical monitoring of the enzymatic activity of nonspecific esterases of viable cells, through the hydrolysis of 1-naphthyl acetate to 1-naphthol. The proposed sensor demonstrated strong correlation (r = 0.979) with viable cell numbers. Furthermore, the model intestinal toxicants diclofenac (DFC, pharmaceutical), okadaic acid (OA, food-safety), and mancozeb (MZB, environmental) were used for the functional evaluation of the proposed sensor using 2D and 3D culture formats. Sensor performance showed high consistency with conventional cell viability/cytotoxicity assays (MTT/CFDA-AM) for all toxicants, with the sensor IC50 values matching the relevant viability LC50 values at the 95% confidence interval range for 2D (DCF: 1.19–1.26 mM, MZB: 10.28–14.18 μM, OA: 40.91–77.13 nM) and 3D culture formats (DCF: 1.02–4.78 mM, MZB: 11.26–15.16 μM, OA: 162.09–179.67 nM). The presented results demonstrate the feasibility of the proposed sensor as a robust endpoint screening tool for both 2D and 3D cytotoxicity assessment

The role of motion management and position verification in lymphoma radiotherapy

In the last decades, the substantial technical progress in radiation oncology offered the opportunity for more accurate planning and delivery of treatment. At the same time, the evolution of systemic treatment and the advent of modern diagnostic tools allowed for more accurate staging and consequently a safe reduction of radiotherapy (RT) target volumes and RT doses in the treatment of lymphomas. As a result, incidental irradiation of organs at risk was reduced, with a consequent reduction of severe late toxicity in long-term lymphoma survivors. Nevertheless, these innovations warrant that professionals pay attention to concurrently ensure precise planning and dose delivery to the target volume and safe sparing of the organs at risk. In particular, target and organ motion should be carefully managed in order to prevent any compromise of treatment efficacy. Several aspects should be taken into account during the treatment pathway to minimise uncertainties and to apply a valuable motion management strategy, when needed. These include: reliable image registration between diagnostic and planning radiologic exams to facilitate the contouring process, image guidance to limit positioning uncertainties and to ensure the accuracy of dose delivery and management of lung motion through procedures of respiratory gating and breath control. In this review, we will cover the current clinical approaches to minimise these uncertainties in patients treated with modern RT techniques, with a particular focus on mediastinal lymphoma. In addition, since uncertainties have a different impact on the dose deposition of protons compared to conventional x-rays, the role of motion management and position verification in proton beam therapy (PBT) will be discussed in a separate section

Proton therapy for adults with mediastinal lymphomas: The international lymphoma radiation oncology group guidelines

Among adult lymphoma survivors, radiation treatment techniques that increase the excess radiation dose to organs at risk (OARs) put patients at risk for increased side effects, especially late toxicities. Minimizing radiation to OARs in adults patients with Hodgkin and non-Hodgkin lymphomas involving the mediastinum is the deciding factor for the choice of treatment modality. Proton therapy may help to reduce the radiation dose to the OARs and reduce toxicities, especially the risks for cardiac morbidity and second cancers. Becauseproton therapymay have some disadvantages, identifying the patients and the circumstances that may benefit the most from proton therapy is important. We present modern guidelines to identify adult lymphoma patients who may derive the greatest benefit from proton therapy, along with an analysis of the advantages and disadvantages of proton treatment. (Blood. 2018;132(16):1635-1646)

Mammalian cell culture for production of recombinant proteins: A review of the critical steps in their biomanufacturing

The manufacturing of recombinant protein is traditionally undertaken in mammalian cell culture. Today, speed, cost and safety are the primary considerations for process improvements in both upstream and downstream manufacturing. Leaders in the biopharmaceutical industry are striving for continuous improvements to increase throughput, lower costs and produce safer more efficacious drugs. This can be achieved through advances in cell line engineering, process development of cell culture, development of chemically defined media and increased emphasis on product characterization. In the first part, this review provides a historical perspective on approved biotherapeutics by regulatory bodies which pave the way for next-generation products (including gene therapy). In the second part, it focuses on the application of in vitro and in vivo cell line engineering approaches, modern process development improvements including continuous manufacturing, recent developments in media formulation, and improvements in critical quality attribute determinations for products produced predominantly in mammalian cells

Assessment of dosimetric errors induced by deformable image registration methods in 4D pencil beam scanned proton treatment planning for liver tumours

PURPOSE: Respiratory impacts in pencil beam scanned proton therapy (PBS-PT) are accounted by extensive 4D dose calculations, where deformable image registration (DIR) is necessary for estimating deformation vector fields (DVFs). We aim here to evaluate the dosimetric errors induced by different DIR algorithms in their resulting 4D dose calculations by using ground truth(GT)-DVFs from 4DMRI. MATERIALS AND METHODS: Six DIR methods: ANACONDA, Morfeus, B-splines, Demons, CT Deformable, and Total Variation, were respectively applied to nine 4DCT-MRI liver data sets. The derived DVFs were then used as input for 4D dose calculation. The DIR induced dosimetric error was assessed by individually comparing the resultant 4D dose distributions to those obtained with GT-DVFs. Both single-/three-field plans and single/rescanned strategies were investigated. RESULTS: Differences in 4D dose distributions among different DIR algorithms, and compared to the results using GT-DVFs, were pronounced. Up to 40 % of clinically relevant dose calculation points showed dose differences of 10 % or more between the GT. Differences in V95(CTV) reached up to 11.34 ± 12.57 %. The dosimetric errors became in general less substantial when applying multiple-field plans or using rescanning. CONCLUSION: Intrinsic geometric errors by DIR can influence the clinical evaluation of liver 4D PBS-PT plans. We recommend the use of an error bar for correctly interpreting individual 4D dose distributions

Evidence-based Review on the Use of Proton Therapy in Lymphoma From the Particle Therapy Cooperative Group (PTCOG) Lymphoma Subcommittee.

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