Analysis of Preplatelets and Their Barbell Platelet Derivatives by Imaging Flow Cytometry

Circulating large ″preplatelets″ undergo fission via barbell platelet intermediates into two smaller, mature platelets. In this study, we determine whether preplatelets and/or barbells are equivalent to reticulated/immature platelets by using ImageStream flow cytometry (ISFC) and super-resolution microscopy. Immature platelets, preplatelets and barbells were quantified in healthy and thrombocytopenic mice, healthy human volunteers, and patients with immune thrombocytopenia (ITP) or undergoing chemotherapy. Preplatelets and barbells were 1.9%±0.18/1.7%±0.48 (n=6) and 3.3%-+1.6/0.5%±0.27 (n=12) of total platelet counts in murine and human whole blood, respectively. Both preplatelets and barbells exhibited high expression of HLA-I with high thiazole orange and mitotracker fluorescence. Tracking dye experiments confirmed that preplatelets transform into barbells and undergo fission ex vivo to increase platelet counts, with dependence upon the cytoskeleton and normal mitochondrial respiration. Samples from antibody-induced thrombocytopenia in mice and patients with ITP had increased levels of both preplatelets and barbells correlating with immature platelet levels. Furthermore, barbells were absent post-chemotherapy in patients. In mice, in vivo biotinylation confirmed that barbells, but not all large platelets, were immature. This study demonstrates that a subpopulation of large platelets are immature preplatelets that can transform into barbells and undergo fission during maturation.

Polycationic Monomeric and Homodimeric Asymmetric Monomethine Cyanine Dyes with Hydroxypropyl Functionality—Strong Affinity Nucleic Acids Binders

New analogs of the commercial asymmetric monomethine cyanine dyes thiazole orange (TO) and thiazole orange homodimer (TOTO) with hydroxypropyl functionality were synthesized and their properties in the presence of different nucleic acids were studied. The novel compounds showed strong, micromolar and submicromolar affinities to all examined DNA ds-polynucleotides and poly rA–poly rU. The compounds studied showed selectivity towards GC-DNA base pairs over AT-DNA, which included both binding affinity and a strong fluorescence response. CD titrations showed aggregation along the polynucleotide with well-defined supramolecular chirality. The single dipyridinium-bridged dimer showed intercalation at low dye-DNA/RNA ratios. All new cyanine dyes showed potent micromolar antiproliferative activity against cancer cell lines, making them promising theranostic agents.

Rhodamine 6G-Ligand Influencing G-Quadruplex Stability and Topology

The involvement of G-quadruplex (G4) structures in nucleic acids in various molecular processes in cells such as replication, gene-pausing, the expression of crucial cancer-related genes and DNA damage repair is well known. The compounds targeting G4 usually bind directly to the G4 structure, but some ligands can also facilitate the G4 folding of unfolded G-rich sequences and stabilize them even without the presence of monovalent ions such as sodium or potassium. Interestingly, some G4-ligand complexes can show a clear induced CD signal, a feature which is indirect proof of the ligand interaction. Based on the dichroic spectral profile it is not only possible to confirm the presence of a G4 structure but also to determine its topology. In this study we examine the potential of the commercially available Rhodamine 6G (RhG) as a G4 ligand. RhG tends to convert antiparallel G4 structures to parallel forms in a manner similar to that of Thiazole Orange. Our results confirm the very high selectivity of this ligand to the G4 structure. Moreover, the parallel topology of G4 can be verified unambiguously based on the specific induced CD profile of the G4-RhG complex. This feature has been verified on more than 50 different DNA sequences forming various non-canonical structural motifs.

Broad Applications of Thiazole Orange in Fluorescent Sensing of Biomolecules and Ions

Fluorescent sensing of biomolecules has served as a revolutionary tool for studying and better understanding various biological systems. Therefore, it has become increasingly important to identify fluorescent building blocks that can be easily converted into sensing probes, which can detect specific targets with increasing sensitivity and accuracy. Over the past 30 years, thiazole orange (TO) has garnered great attention due to its low fluorescence background signal and remarkable ‘turn-on’ fluorescence response, being controlled only by its intramolecular torsional movement. These features have led to the development of numerous molecular probes that apply TO in order to sense a variety of biomolecules and metal ions. Here, we highlight the tremendous progress made in the field of TO-based sensors and demonstrate the different strategies that have enabled TO to evolve into a versatile dye for monitoring a collection of biomolecules.