Inhibition of Firefly Luciferase by General Anesthetics: Effect on In Vitro and In Vivo Bioluminescence Imaging

<div><h3></h3><p>Bioluminescence imaging is routinely performed in anesthetized mice. Often isoflurane anesthesia is used because of its ease of use and fast induction/recovery. However, general anesthetics have been described as important inhibitors of the luciferase enzyme reaction.</p> <h3>Aim</h3><p>To investigate frequently used mouse anesthetics for their direct effect on the luciferase reaction, both in vitro and in vivo.</p> <h3>Materials and Methods</h3><p>isoflurane, sevoflurane, desflurane, ketamine, xylazine, medetomidine, pentobarbital and avertin were tested in vitro on luciferase-expressing intact cells, and for non-volatile anesthetics on intact cells and cell lysates. In vivo, isoflurane was compared to unanesthetized animals and different anesthetics. Differences in maximal photon emission and time-to-peak photon emission were analyzed.</p> <h3>Results</h3><p>All volatile anesthetics showed a clear inhibitory effect on the luciferase activity of 50% at physiological concentrations. Avertin had a stronger inhibitory effect of 80%. For ketamine and xylazine, increased photon emission was observed in intact cells, but this was not present in cell lysate assays, and was most likely due to cell toxicity and increased cell membrane permeability. In vivo, the highest signal intensities were measured in unanesthetized mice and pentobarbital anesthetized mice, followed by avertin. Isoflurane and ketamine/medetomidine anesthetized mice showed the lowest photon emission (40% of unanesthetized), with significantly longer time-to-peak than unanesthetized, pentobarbital or avertin-anesthetized mice. We conclude that, although strong inhibitory effects of anesthetics are present in vitro, their effect on in vivo BLI quantification is mainly due to their hemodynamic effects on mice and only to a lesser extent due to the direct inhibitory effect.</p> </div

Virological surveillance report of the NRC influenza for season 2020-2021

Amidst the COVID-19 pandemic, the sentinel surveillance resumed slowly in the season 2020-2021. Only the severe acute respiratory infections (SARI) allowed to properly evaluate the circulation of respiratory viruses during the 2020-2021 season. The season was characterized by the absence of an influenza virus epidemic, and the return of parainfluenza viruses and respiratory syncytial viruses detected mainly in children. SARS-CoV-2 continued to intensively circulate and was mainly detected in adults and older adults. Other respiratory viruses such as metapneumoviruses, seasonal coronaviruses, rhino- and enteroviruses, and adenoviruses were also detected, without clear epidemic&nbsp;wave.</p

Virological surveillance of influenza in Belgium, season 2019-2020

The 2019-2020 winter season was characterized by the occurrence after the flu epidemic of the COVID-19 pandemic.&nbsp; The Influenza epidemic in Belgium lasted 6 weeks and was a flu season of moderate intensity characterized by the co-circulation of A(H1N1)pdm09 and A(H3N2), with the predominance of A(H1N1). The epidemic threshold was crossed at &nbsp;week 4-2020 (January 13 to January 19, 2020 with an incidence of 245 consultations /100.000 inhabitants and the peak was reached in week 5 with 550 consultations/100.000 inhabitants. After week 5- 2020, the incidence of ILI consultations decreased but remained above the threshold for several weeks likely due to the COVID-19 epidemic with a new ILI peak &nbsp;at week 13 exceeding the influenza peak seen in &nbsp;week 5 &nbsp;(Fig. 1). The emergence of COVID-19, spreading through respiratory transmission, required the implementation of physical distancing measures likely contributed to an abrupt decline of the influenza&nbsp;season. The majority of the H1N1 viruses fell in the 6B.1A5A subgroup represented by the reference strain&nbsp;A/Norway/3433/2018. About half of the sequenced A(H3N2) viruses belonged to the clade 3C.2a1 and the remaining belonged to the &nbsp;clade 3C.3a close the vaccine strain for the northern hemisphere&nbsp;A/Kansas/14/2017. Most of &nbsp;the seqenced influenza B-Victoria viruses were triple-deletion variants similar to B/Washington/02/2019.&nbsp;&nbsp; Respiratory &nbsp;samples were also analysed for other respiratory viruses. In the ILI population, &nbsp;70 % of the patients were positive for at least one respiratory virus (including Influenza and co-infections). In the &nbsp;SARI population, 52% of the patients were positive for at least one respiratory viruses (including influenza, SARS-COV-2, other respiratory viruses&nbsp; or different combination of co-infection). From week 10 , the first SARS-CoV-2 patient were&nbsp;diagnosed. These patients were mostly adults and children above 14 years&nbsp;old. Severity was moderate in comparison to the previous season and comparable to previous&nbsp;seasons. None of the analyzed strains presented mutations known to be associated to resistance to antivirals neuraminidase inhibitors (Oseltamivir et&nbsp;Zanamivir).</p

In vivo detection of small tumour lesions by multi-pinhole SPECT applying a (99m)Tc-labelled nanobody targeting the Epidermal Growth Factor Receptor.

The detection of tumours in an early phase of tumour development in combination with the knowledge of expression of tumour markers such as epidermal growth factor receptor (EGFR) is an important prerequisite for clinical decisions. In this study we applied the anti-EGFR nanobody (99m)Tc-D10 for visualizing small tumour lesions with volumes below 100 mm(3) by targeting EGFR in orthotopic human mammary MDA-MB-468 and MDA-MB-231 and subcutaneous human epidermoid A431 carcinoma mouse models. Use of nanobody (99m)Tc-D10 of a size as small as 15.5 kDa enables detection of tumours by single photon emission computed tomography (SPECT) imaging already 45 min post intravenous administration with high tumour uptake (>3% ID/g) in small MDA-MB-468 and A431 tumours, with tumour volumes of 52.5 mm(3) ± 21.2 and 26.6 mm(3) ± 16.7, respectively. Fast blood clearance with a serum half-life of 4.9 min resulted in high in vivo contrast and ex vivo tumour to blood and tissue ratios. In contrast, no accumulation of (99m)Tc-D10 in MDA-MB-231 tumours characterized by a very low expression of EGFR was observed. Here we present specific and high contrast in vivo visualization of small human tumours overexpressing EGFR by preclinical multi-pinhole SPECT shortly after administration of anti-EGFR nanobody (99m)Tc-D10.Open-Access Publikationsfunds 2016peerReviewe

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.We thank FAPESP (BEPE 2015/07509-1 and 2017/13168-8 to B.B., and 2013/25504-1 to A.C.B.B.), Xunta de Galicia (M.J.M.), FCT Portugal (FCT Investigator to G.J.L.B., IF/00624/2015), the EU (Marie Sklodowska-Curie ITN Protein Conjugates, GA 675007, including a PhD Studentship to X.F.), the Ministerio de Economía y Competitividad (projects CTQ2015-67727-R and UNLR13-4E-1931 to F.C. and CTQ2015-70524-R and RYC-2013-14706 to G.J.O.) and the Universidad de La Rioja (FPI Studentship to I.C.). We also thank S. Massa and N. Devoogdt (Vrije Universiteit Brussel (VUM), Brussels) for the generous gift of the Her2-targeting nanobody 2Rb17c, Genentech for providing Thiomab LC-V205C and trastuzumab antibodies, and D. Neri’s laboratory (Swiss Federal Institute of Technology (ETH Zürich), Zurich) for the generous gift of the F16 antibody. G.J.L.B. is a Royal Society University Research Fellow (UF110046 and URF/R/180019) and the recipient of a European Research Council Starting Grant (TagIt, GA 676832)