Comparison of standardised versus non-standardised methods for testing the in vitro potency of oxytetracycline against mannheimia haemolytica and pasteurella multocida

The in vitro pharmacodynamics of oxytetracycline was established for six isolates of each of the calf pneumonia pathogens Mannheimia haemolytica and Pasteurella multocida. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and bacterial time-kill curves were determined in two matrices, Mueller Hinton broth (MHB) and calf serum. Geometric mean MIC ratios, serum:MHB, were 25.2:1 (M. haemolytica) and 27.4:1 (P. multocida). The degree of binding of oxytetracycline to serum protein was 52.4%. Differences between serum and broth MICs could not be accounted for by oxytetracycline binding to serum protein. In vitro time-kill data suggested a co-dependent killing action of oxytetracycline. The in vitro data indicate inhibition of the killing action of oxytetracycline by serum factor(s). The nature of the inhibition requires further study. The outcome of treatment with oxytetracycline of respiratory tract infections in calves caused by M. haemolytica and P. multocida may not be related solely to a direct killing action

Pharmacokinetic–pharmacodynamic integration and modelling of oxytetracycline for the calf pathogens Mannheimia haemolytica and Pasteurella multocida

A calf tissue cage model was used to study the pharmacokinetics (PK) and pharmacodynamics (PD) of oxytetracycline in serum, inflamed (exudate) and noninflamed (transudate) tissue cage fluids. After intramuscular administration, the PK was characterized by a long mean residence time of 28.3 hr. Based on minimum inhibitory concentrations (MICs) for six isolates each of Mannheimia haemolytica and Pasteurella multocida, measured in serum, integration of in vivo PK and in vitro PD data established area under serum concentration–time curve (AUC0–∞)/MIC ratios of 30.0 and 24.3 hr for M. haemolytica and P. multocida, respectively. Corresponding AUC0–∞/MIC ratios based on MICs in broth were 656 and 745 hr, respectively. PK-PD modelling of in vitro bacterial time–kill curves for oxytetracycline in serum established mean AUC0–24 hr/MIC ratios for 3log10 decrease in bacterial count of 27.5 hr (M. haemolytica) and 60.9 hr (P. multocida). Monte Carlo simulations predicted target attainment rate (TAR) dosages. Based on the potency of oxytetracycline in serum, the predicted 50% TAR single doses required to achieve a bacteriostatic action covering 48-hr periods were 197 mg/kg (M. haemolytica) and 314 mg/kg (P. multocida), respectively, against susceptible populations. Dosages based on the potency of oxytetracycline in broth were 25- and 27-fold lower (7.8 and 11.5 mg/kg) for M. haemolytica and P. multocida, respectively

Pharmacokinetic/pharmacodynamic integration and modelling of oxytetracycline for the porcine pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida

Pharmacokinetic–pharmacodynamic (PK/PD) integration and modelling were used to predict dosage schedules of oxytetracycline for two pig pneumonia pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) were determined in broth and porcine serum. PK/PD integration established ratios of average concentration over 48 h (Cav0–48 h)/MIC of 5.87 and 0.27 µg/mL (P. multocida) and 0.70 and 0.85 µg/mL (A. pleuropneumoniae) for broth and serum MICs, respectively. PK/PD modelling of in vitro time–kill curves established broth and serum breakpoint values for area under curve (AUC0–24 h)/MIC for three levels of inhibition of growth, bacteriostasis and 3 and 4 log10 reductions in bacterial count. Doses were then predicted for each pathogen, based on Monte Carlo simulations, for: (i) bacteriostatic and bactericidal levels of kill; (ii) 50% and 90% target attainment rates (TAR); and (iii) single dosing and daily dosing at steady-state. For 90% TAR, predicted daily doses at steady-state for bactericidal actions were 1123 mg/kg (P. multocida) and 43 mg/kg (A. pleuropneumoniae) based on serum MICs. Lower TARs were predicted from broth MIC data; corresponding dose estimates were 95 mg/kg (P. multocida) and 34 mg/kg (A. pleuropneumoniae)

Electrochemical Biosensing in Cancer Diagnostics and Follow-up

In cancer, screening and early detection are critical for the success of the patient's treatment and to increase the survival rate. The development of analytical tools for non‐invasive detection, through the analysis of cancer biomarkers, is imperative for disease diagnosis, treatment and follow‐up. Tumour biomarkers refer to substances or processes that, in clinical settings, are indicative of the presence of cancer in the body. These biomarkers can be detected using biosensors, that, because of their fast, accurate and point of care applicability, are prominent alternatives to the traditional methods. Moreover, the constant innovations in the biosensing field improve the determination of normal and/or elevated levels of tumour biomarkers in patients’ biological fluids (such as serum, plasma, whole blood, urine, etc.). Although several biomarkers (DNA, RNA, proteins, cells) are known, the detection of proteins and circulating tumour cells (CTCs) are the most commonly reported due to their approval as tumour biomarkers by the specialized entities and commonly accepted for diagnosis by medical and clinical teams. Therefore, electrochemical immunosensors and cytosensors are vastly described in this review, because of their fast, simple and accurate detection, the low sample volumes required, and the excellent limits of detection obtained. The biosensing strategies reported for the six most commonly diagnosed cancers (lung, breast, colorectal, prostate, liver and stomach) are summarized and the distinct phases of the sensors’ constructions (surface modification, antibody immobilization, immunochemical interactions, detection approach) and applications are discussed.Maria Freitas is grateful to FCT‐Fundação para a Ciência e a Tecnologia for her PhD grant (SFRH/BD/111942/2015), financed by POPH‐QREN‐Tipologia 4.1‐Formaçãpo Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT) through project UID/QUI/50006/2013.info:eu-repo/semantics/publishedVersio

Tracking Arachis hypogaea Allergen in Pre-Packaged Foodstuff: A Nanodiamond-Based Electrochemical Biosensing Approach

The present work reports a nanodiamond-based voltammetric immunosensing platform for the analysis of a food allergen (Ara h 1) present in peanuts (Arachis hypogaea). The possibility of the usage of nanodiamonds (d = 11.2 ± 0.9 nm) on screen-printed carbon electrodes (SPCE/ND) in a single-use two-monoclonal antibody sandwich assay was studied. An enhanced electroactive area (~18%) was obtained and the biomolecule binding ability was improved when the 3D carbon-based nanomaterial was used. The antibody-antigen interaction was recognized through the combination of alkaline phosphatase with 3-indoxyl phosphate and silver ions. Linear Sweep Voltammetry (LSV) was applied for fast signal acquisition and scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) support the voltammetric approach and confirm the presence of silver particles on the electrode surface. The proposed immunosensor provided a low limit of detection (0.78 ng·mL−1) and highly precise (RSD < 7.5%) and accurate results. Quantification of Ara h 1 in commercial foodstuffs (e.g., crackers, cookies, protein bars) that refer to the presence of peanuts (even traces) on the product label was successfully achieved. The obtained data were in accordance with recovery results (peanut addition, %) and the foodstuff label. Products with the preventive indication “may contain traces” revealed the presence of peanuts lower than 0.1% (m/m). The method’s results were validated by comparison with an enzyme-linked immunosorbent assay. This allows confident information about the presence of allergens (even at trace levels) that leads to profitable conditions for both industry and consumers.This research received financial support from the European Union (FEDER funds through COMPETE POCI-01-0145-FEDER-030735) and National Funds (Fundação para a Ciência e a Tecnologia (FCT)) through the project PTDC/QUI-QAN/30735/2017—TracAllerSens—Electrochemical sensors for the detection and quantification of trace amounts of allergens in food products.info:eu-repo/semantics/publishedVersio

Evaluation of a Bacillus stearothermophilus tube test as a screening tool for anticoccidial residues in poultry

A Bacillus stearothermophilus var. calidolactis C953 tube test was evaluated for its ability in detecting the residue of selected anticoccidial drugs in poultry, specically sulfamethazine, furazolidone, and amprolium. Various concentrations of each drug were injected into chicken liver and kidney tissues and these tissues were tested to determine the drug detection limits for each drug. The detection limit was defined as the drug concentration at which 95% of the test results were interpreted as positive. The limits of detection in liver tissue were 0.35 µg/ml for furazolidone, 0.70 µg/ml for sulfamethazine and 7.80 µg/ml for amprolium. In kidney tissues, they were 0.30 µg/ml for furazolidone, 0.54 µg/ml for sulfamethazine, and 7.6 µg/ml for amprolium. It was concluded that this tube test could be used to screen for the residue of these three drugs in poultry

Tracking a Major Egg Allergen to Assess Commercial Food Label Compliance: Towards a Simple and Fast Immunosensing Device

An amperometric immunosensor was developed for the analysis of the major egg-white allergen ovotransferrin (Gal d 3) in commercial food products because the (accidental) intake, skin contact with, and/or inhalation of eggs can lead to severe disorders in allergic individuals. Employing a sandwich-type immunosensing strategy, screen-printed carbon electrodes (SPCE) were biomodified with anti-Gal d 3 (capture) antibodies, and the allergen’s detection was achieved with anti-Gal d 3 antibodies labelled with horseradish peroxidase (HRP). The 3,3′,5,5′-tetramethylbenzidine (TMB)/H2O2 reaction with HRP was used to obtain the electrochemical (amperometric) signal. An attractive assay time of 30 min and a remarkable analytical performance was achieved. The quantification range was established between 55 and 1000 ng·mL−1, with a limit of detection of 16 ng·mL−1. The developed method demonstrated good precision (Vx0 = 5.5%) and provided precise results (CV < 6%). The sensor also detected extremely low amounts (down to 0.010%) of egg. The analysis of seven raw and/or cooked egg and egg-white samples indicated that food processing influences the amount of allergen. Furthermore, to assure the compliance of product labelling with EU legislation, 25 commercial food ingredients/products were analysed. The accuracy of the results was confirmed through an ELISA assay. The stability of the ready-to-use sensing surface for 20 days allows a reduction of the reagents’ volumes and cost.This research received financial support from the European Union (FEDER funds through COMPETE POCI-01-0145-FEDER-030735) and National Funds (Fundação para a Ciência e a Tecnologia (FCT)) through the project PTDC/QUI-QAN/30735/2017—TracAllerSens—Electrochemical sensors for the detection and quantification of trace amounts of allergens in food products.info:eu-repo/semantics/publishedVersio

High-performance electrochemical immunomagnetic assay for breast cancer analysis

Despite the evolution of targeted therapies in oncology, some challenges such as screening and early diagnosis of cancer-related biomarkers still remain. The analysis of the Human Epidermal growth factor Receptor 2 (HER2) in biological fluids provides essential information for effective treatments. In this work we report the development of an electrochemical immunomagnetic bioassay for the analysis of the extracellular domain of HER2 (HER2-ECD) in human serum and cancer cells. Biomodified carboxylic acid functionalized magnetic beads (COOH-MBs) were used as the capture probe and an antibody labelled with alkaline phosphatase (AP) as the signalling probe. In the presence of HER2-ECD a sandwich complex was formed on the MBs, which were magnetically attracted to the surface of a screen-printed carbon electrode (SPCE). After the addition of 3-indoxyl phosphate and silver ions, used as the enzymatic substrate, the immunological interaction was detected by linear sweep voltammetry. Two linear concentration ranges were established: one between 5.0 and 50 ng/mL and another between 50 and 100 ng/mL. The developed assay provided a clinically useful detection limit (2.8 ng/mL) and has an adequate precision (Vx0 < 5%). The assay provided accurate results and was selective towards the target biomarker. Additionally, CTCs were analysed in human serum and a detection limit of 3 cells/mL was achieved for the HER+ breast cancer cell line SK-BR-3.The authors are grateful for the financial support from the Fundação para a Ciência e a Tecnologia (FCT) / the Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through national funds (Portugal) (LAQV - UID/QUI/50006/2019 and CINTESIS - UID/IC/4255/2019). Maria Freitas is grateful to FCT for her PhD grant (SFRH/BD/111942/2015), financed by POPH- QREN-Tipologia 4.1-Formação Avançada, subsidized by Fundo Social Europeu and the MCTES. The authors are also thankful to Rui Rocha and CEMUP “Centro de Materiais da Universidade do Porto” for the SEM workinfo:eu-repo/semantics/publishedVersio

Breast cancer biomarker (HER2-ECD) detection using a molecularly imprinted electrochemical sensor

The extracellular domain of the human epidermal growth factor receptor 2 (HER2-ECD) is a protein breast cancer biomarker. Its quantification in peripheral blood could provide an important contribution to diagnostics and patient follow-up. In this work an electrochemical molecularly imprinted polymer (MIP) sensor for the quantification of HER2-ECD was developed. The MIP was electropolymerized by cyclic voltammetry using a solution containing phenol and HER2-ECD on a screen-printed gold electrode (AuSPE). The sensor was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The analysis of HER2-ECD was performed by differential pulse voltammetry using ([Fe(CN)6]3−/4−as redox probe. The linear range was established in the concentration interval from 10 to 70 ng/mL HER2-ECD, with a limit of detection of 1.6 ng/L and a limit of quantification of 5.2 ng/mL. Through the analysis of other protein biomarkers, the MIP sensor was found to be selective. Furthermore, these proteins did not interfere in the analysis of the selected biomarker. The developed sensor was used for the analysis of spiked human serum samples, providing adequate recovery values and precise results. The outcomes of this study indicate that the developed MIP sensor could be useful in the non-invasive analysis of HER2-ECD in breast cancer patients.Maria Freitas is grateful to FCT (Fundação para a Ciência e a Tecnologia) for her PhD grant (SFRH/BD/111942/2015). Patrícia Rebelo is grateful to FCT (Fundação para a Ciência e a Tecnologia) for her PhD grant (SFRH/BD/132384/2017). João Pacheco is grateful to FCT for his Postdoc. grant (SFRH/BPD/101419/2014), financed by POPH-QREN-Tipologia 4.1-Formação Avançada, subsidized by Fundo Social Europeu and Ministério da Ciência, Tecnologia e Ensino Superior. This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT) through project UID/QUI/50006/2013.info:eu-repo/semantics/publishedVersio

Impact of magnetite nanoparticles on the syntrophic dechlorination of 1,2-dichloroethane

In anaerobic environments microorganisms exchange electrons with community members and with soil and groundwater compounds. Interspecies electron transfer (IET) occurs by several mechanisms: diffusion of redox compounds or direct contact between cells. This latter mechanism may be facilitated by the presence of conductive nanoparticles (NP), possibly serving as electron conduits among microorganisms. Our study examined the effect of magnetite (Fe3O4) NP on the dechlorination of 1,2-dichloroethane (1,2-DCA) by a mixed-culture. The addition of NP (170 mg L− 1 total Fe) enhanced the acetate-driven reductive dechlorination of 1,2-DCA to harmless ethene (via reductive dihaloelimination) up to 3.3-times (2.3 μeq L− 1 d− 1 vs. 0.7 μeq L− 1 d− 1), while decreasing the lag time by 0.8 times (23 days) when compared to unamended (magnetite-free) microcosms. Dechlorination activity was correlated with the abundance of Dehalococcoides mccartyi, which accounted up to 50% of total bacteria as quantified by CARD-FISH analysis, pointing to a key role of this microorganism in the process. Given the widespread abundance of conductive minerals in the environment, the results of this study may provide new insights into the fate of 1,2-DCA and suggest new tools for its remediation by linking biogeochemical mechanisms.The authors would like to thank FCT (Portuguese Foundation for Science and Technology) for the financial support of Patrícia Leitão through the PhD grant SFRH/BD/87312/2012 and to CEMUP - Centro de Materiais da Universidade do Porto for expert assistance and helpful discussions about SEM measurements.info:eu-repo/semantics/publishedVersio