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Internalization of Lipopolysaccharide by Phagocytes
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
Richard L. Kitchens, Robert S. Munford
More recently, several groups have used laser confocal microscopy to follow the intracellular movement of LPS tagged with fluorescent adjuncts such as FITC or BODIPY (1,2,22,30,59). These studies have confirmed that monocytes internalize LPS aggregates rapidly, so that discrete pockets of bright fluorescence become discernible within 5 minutes after LPS is added (22,30,59). BODIPY-LPS that was presented to cells as monomeric LPS-sCD14 complexes was internalized slowly (consistent with the slow internalization of monomeric [3H]LPS by THP-1 cells (22), but it also accumulated in discrete intracellular locations (1,2). The nature of the intracellular vesicles has not been determined.
Molecular Imaging of Viable Cancer Cells
Published in Shoogo Ueno, Bioimaging, 2020
As a cancer-targeting antibody, we selected a monoclonal antibody directed toward human epidermal growth factor receptor type 2 (HER2), trastuzumab, which is internalized via endocytosis after binding to HER2. As the small-molecular fluorophore, we newly designed a probe whose fluorescence is activated at acidic pH; thus, after cellular internalization, the probe is activated by the low pH (pH 5.0–6.0) of the lysosome. We developed a pH-sensitive fluorescence probe, DiEtN-BDP, by incorporating N,N-diethylaniline as the pH-responsive PeT donor into the BODIPY fluorophore, and used it to label trastuzumab, obtaining a probe-antibody conjugate, DiEtN-BDP–trastuzumab. This conjugate is almost non-fluorescent at neutral pH due to PeT quenching from the aniline moiety to the fluorophore, but becomes highly fluorescent at acidic pH, showing a substantial increase in fluorescence emission. We then evaluated the performance of this conjugate by applying it for ex vivo fluorescence imaging of freshly resected lungs bearing metastatic genetically engineered NIH3T3 mouse fibroblast tumors overexpressing HER2 receptors. The probe produced signals only from HER2-positive tumors, and the background fluorescence from normal lung or heart tissue was well suppressed, resulting in a high TBR ratio.39
Detection Techniques for Single Nucleotide Polymorphisms
Published in Attila Lorincz, Nucleic Acid Testing for Human Disease, 2016
W. Mathias Howell, Johan Stenberg, Chatarina Larsson, Mats Nilsson, Ulf Landegren
Many fluorescent detection reagents have been developed to stain or tag DNA molecules for SNP genotyping. For example, SYBR® Green I from Molecular Probes (Eugene, Oregon, USA, http://probes.invitrogen.com/) is an intercalating dye that undergoes a 10,000-fold increase in fluorescence when bound to double-stranded DNA. This dye has proven useful in assays that detect the presence or absence of DNA duplexes as part of a genotyping procedure.76 Dyes with other desirable characteristics such as a large Stokes shift (difference between peak excitation and emission wavelengths), a high extinction coefficient (ability to capture excitation photons), and a high quantum yield (efficiency of converting between incoming and outgoing photons) are under continual development, exemplified by the Bodipy® and Alexa Flour® dye series also from Molecular Probes. Quantum dots or qdots represent a relatively new class of fluorescent labels composed of Zn or Cd nanoparticles encapsulated in a biocompatible, hydrophilic shell. Among the advantages of qdots over other fluorophores is that they are highly resistant to photobleaching (fluorophore destruction due to over-excitation), have relatively narrow emission spectra, and can all be excited at a fixed wavelength of light.
Polymerization-sensitive switch-on monomer for terminal transferase activity assay
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Bhagwan S. Batule, Chang Yeol Lee, Ki Soo Park, Hyun Gyu Park
To assess the sensitivity of the proposed strategy, the fluorescence intensities at 516 nm were measured as a function of TdT concentration. As shown in Figure 3, the linear relationship is observed in the range from 0 to 50 U/mL (R2=0.9942, F516 = 1141.4 × CTdT/U mL−1 + 3416.9, where CTdT is the concentration of TdT) with the detection limit of 0.64 U/mL (3σ/slope), a value that is comparable or superior to those associated with other fluorescence-based TdT assays [20–24]. The selectivity was also determined by employing other enzymes such as exonucleases, ligases and template-dependent DNA polymerases. The results in Figure 4 show that the fluorescence of BODIPY is efficiently switched on by the action of TdT, while other enzymes induce the negligible fluorescence enhancement even though they are present at 10 times higher concentration than that of TdT, confirming that it has high selectivity toward TdT.
Effects of boron-containing compounds on immune responses: review and patenting trends
Published in Expert Opinion on Therapeutic Patents, 2019
Karla S. Romero-Aguilar, Ivonne M. Arciniega-Martínez, Eunice D. Farfán-García, Rafael Campos-Rodríguez, Aldo A. Reséndiz-Albor, Marvin A. Soriano-Ursúa
BCCs have been shown to act on inflammatory processes. Borax (3) has some effects on the inflammatory processes induced by herpesvirus inoculation [93]. In addition, boric acid (1) altered cytokine profiles in mice infected with Nematoda [94]; benzoxaboroles (4) (including some available for human use such as tavaborole and crisaborole) [95] and some boron-clusters exert double activity as antibiotic and anti-inflammatory modulators [96]. Boron-dipyrromethenes, also known as BODIPY (18) compounds, another BCC group originally designed as biomarkers, exhibit some antibiotic and anti-inflammatory activities [97]. Vaborbactam (19), a beta-lactamase inhibitor based on a pharmacophore of highly active cyclic boronic acid, is currently used as an antibiotic, and evaluation of this inhibitor and other BCCs has revealed some anti-inflammatory effects [87,98].
Lipid membranes accelerate amyloid formation in the mouse model of AA amyloidosis
Published in Amyloid, 2019
Aida Vahdat shariat panahi, Per Hultman, Karin Öllinger, Gunilla T. Westermark, Katarzyna Lundmark
Animals were anesthetized with isoflurane and decapitated. Blood was collected, and serum was recovered by centrifugation at 2000 × g for 10 min. Blood smears were made from mice treated with BODIPY-Lipo. Spleen and a part of the liver were placed in Tissue-Tek OCT compound (Sacura, Zoeterwoude, Netherlands) and snap frozen. In addition, small pieces of spleen were collected for Western blot analysis. Samples were stored at −80 °C awaiting analysis.