Thiazole Orange Research Articles

Tuning the selectivity of a commercial cyanine nucleic acid dye for preferential sensing of hybrid telomeric G-quadruplex DNA

Recent years have witnessed an ever-increasing interest in G-quadruplexes for diagnostic and therapeutic applications. This calls for the development of highly selective probes that can differentiate a particular structure among large quantities of G-quadruplexes. However, to this day, there are few probes that show satisfactory selectivity for a particular G-quadruplex structure. In this study, we engineered a new probe called TO-BTZ by introducing an additional benzothiazole moiety on a commercial cyanine dye thiazole orange (TO). Such a modification of TO significantly tuned its selectivity toward the hybrid telomeric G-quadruplex DNA by specifically targeting the unique binding site formed near the 5′-end G-quartet. Such specific interactions disassembled the TO-BTZ H-aggregate to its monomers and further hindered the molecular rotation of the TO-BTZ monomer, thus leading to fluorescence enhancement. Furthermore, we observed that TO-BTZ has the potential to sense telomeric G-quadruplexes in cells. Taken together, this work will shed light on the search for a new generation of probes that can distinguish between different G-quadruplex structures.

Investigating Binding Mechanisms of Small Molecules to Quadruplex DNA

Guanine quadruplexes (G4) are dynamic structures found naturally in guanine-rich DNA. When folded, they have been found to block the expression of genes related to diseases including cancer1 and are therefore of significant interest in the field of biomedical research. Our research group has developed a family of chemical binders which strongly stabilize folded G4. This project focuses on quantifying the interaction of these binders with various quadruplexes, through their binding constant (a measure of relative preference for the bound vs unbound state). This constants can be determined through the use of a Fluorescence Intercalation Displacement (FID) biophysical assay,2 a method which measures the competitive binding of the binder molecule and a fluorescent probe to the quadruplex (see figure 1). As a preliminary step, we perform direct titrations of the fluorescent probe, Thiazole Orange (TO), with quadruplex DNA, to determine its binding constant and TO:DNA stoichiometry. TO fluoresces only when bound to a substrate,3 allowing us to track the formation of the TO*DNA complex through an increase in fluorescence. In the FID titration, however, the binder displaces the fluorescent probe, and a decrease in fluorescence intensity is observed. We also discuss mathematical methods used to fit the experimental data in order to determine binding constants for both the fluorescent probe and our binders. A robust model should give evidence in support of the expected binding phenomena.
 References
 1. Hurley, L. et. al. The Febs Journal, 2010, 277, 3459-3469 2. Monchaud, D. et. al. Biochimie, 2008, 90: 1209-1223 3. Yaron, D. et. al. The Journal of Physical Chemistry A. 2008, 112, 9692-9701

Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization.

Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signaling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here, we introduce qFIT (quantitative forced intercalation) probes that allow the detection both of probe-target complexes and of unbound probes on separate, independent channels. A surrogate nucleobase based on thiazole orange (TO) probes the hybridization status. The second channel involves a nonresponsive near-IR dye, which serves as a reporter of concentration. We show that the undesirable perturbation of the hybridization reporter TO is avoided when the near-IR dye Cy7 is connected by means of short triazole linkages in an ≥18 nucleotides distance. We used the qFIT probes to localize and quantify oskar mRNA in fixed egg chambers of wild-type and mutant Drosophila melanogaster by wash-free fluorescence in situ hybridization. The measurements revealed a relative 400-fold enrichment of oskar within a 3000 μm3 large volume at the posterior pole of stage 8-9 oocytes, which peaked at a remarkably high 1.8 μM local concentration inside 0.075 μm3 volume units. We discuss detection limits and show that the number of oskar mRNA molecules per oocyte is independent of the oocyte size, which suggests that the final levels are attained already during the onset of oskar localization at stage 8.

Fluorogenic thiazole orange TOTFO probes stabilise parallel DNA triplexes at pH 7 and above.

The instability of DNA triplexes particularly at neutral pH and above severely limits their applications. Here, we demonstrate that the introduction of a thiazole orange (TO) intercalator onto a thymine nucleobase in triplex forming oligonucleotides (TFOs) resolves this problem. The stabilising effects are additive; multiple TO units produce nanomolar duplex binding and triplex stability can surpass that of the underlying duplex. In one example, a TFO containing three TO units increased the triplex melting temperature at pH 7 by a remarkable 50 °C relative to the unmodified triplex. Notably, TO intercalation promotes TFO binding to target sequences other than pure polypurine tracts by the use of 5-(1-propynyl)cytosine (pC) against C:G inversions. By overcoming the instability of triplexes across a broad range of pH and sequence contexts, these very simple 'TOTFO' probes could expand triplex applications into many areas including diagnostics and cell imaging.

Halogen-containing thiazole orange analogues - new fluorogenic DNA stains.

Novel asymmetric monomeric monomethine cyanine dyes 5a–d, which are analogues of the commercial dsDNA fluorescence binder thiazole orange (TO), have been synthesized. The synthesis was achieved by using a simple, efficient and environmetally benign synthetic procedure to obtain these cationic dyes in good to excellent yields. Interactions of the new derivatives of TO with dsDNA have been investigated by absorption and fluorescence spectroscopy. The longest wavelength absorption bands in the UV–vis spectra of the target compounds are in the range of 509–519 nm and these are characterized by high molar absorptivities (63000–91480 L·mol−1·cm−1). All investigated dyes from the series are either not fluorescent or their fluorescence is quite low, but they become strongly fluorescent after binding to dsDNA. The influence of the substituents attached to the chromophores was investigated by combination of spectroscopic (UV–vis and fluorescence spectroscopy) and theoretical (DFT and TDDFT calculations) methods.

Thiazole Orange as an Alternative to Antibody Binding for Detecting Triple-helical DNA in Heterochromatin of Drosophila and Rhynchosciara.

The standard method for detecting triple-stranded DNA over the last 1.5 decades has been immune detection using antibodies raised against non-canonical nucleic acid structures. Many fluorescent dyes bind differentially to nucleic acids and often exhibit distinctive staining patterns along metaphase chromosomes dependent upon features, including binding to the major and minor DNA grooves, level of chromatin compaction, nucleotide specificity, and level of dye stacking. Relatively recently, the fluorochrome Thiazole Orange (TO) was shown to preferentially bind to triplex DNA in gels. Here, we demonstrate that TO also detects triplex DNA in salivary gland chromosomes of Drosophila melanogaster and Rhynchosciara americana identical in location and specificity to observations using antibodies. This finding may enable triple-stranded DNA investigations to be carried out on a much broader and reproducible scale than hitherto possible using antibodies, where a frequently encountered problem is the difference in detection specificity and sensitivity between one antibody and another.

VPS4A : A Novel Candidate Gene for Congenital Dyserythropoietic Anemia

CDAR (ClinicalTrials.gov Identifier: NCT02964494), a registry for patients with Congenital Dyserythropoietic Anemia (CDA) in North America, was recently created with the goal to provide a longitudinal database and associated biorepository to facilitate natural history studies and research on the molecular pathways involved in the pathogenesis of CDAs. A 2 y.o. female patient with transfusion dependent anemia, pathologic diagnosis of Congenital Dyserythropoietic Anemia type I (CDA-I), and neurodevelopmental delay was enrolled in CDAR. Next Generation sequencing and deletion/duplication assay identified no mutations in the known CDA-associated genes, including CDAN1 and C15orf41, which are causative for CDA-I. Whole-exome sequencing for the patient and her parents (family-trio design) revealed a novel, de novo VPS4A missense variant located in the last codon of exon 8, potentially affecting splicing. VPS4A is an ATPase which, in association with the endosomal sorting complex required for transport (ESCRT), has been shown to play a critical role in cell division of HeLa cells in vitro, concentrating at the spindle poles during mitosis and at the midbody during cytokinesis. The aim of this work is to validate the pathogenetic role of the VPS4A variant for CDA and further investigate the role of VPS4A in erythroblast mitosis and cytokinesis.Central review of the patient's bone marrow aspirate smears revealed bi-nucleated erythroblasts in the range of 3-7%, a criterion compatible with CDA-I. However, cytoplasmic bridges were noted (arrows in Figure 1A) rather than the nuclear bridges typical of CDA-I. Immunofluorescence staining performed on erythroblasts generated ex vivo from normal CD34+ cells verified that VPS4A localizes to the spindle poles during mitosis and the midbody during cytokinesis in dividing human erythroid cells analyzed by Imaging Flow Cytometry (Figure 1B). The level of VPS4A mRNA expression in the patient's reticulocytes was evaluated by qPCR using three different sets of primers and found to be decreased by 55-70% compared to control reticulocytes and knock-down of VPS4A in normal CD34+ cells resulted in erythroid cultures enriched in binucleated cells. Induced pluripotent stem cells (iPSCs) were generated from the patient's peripheral blood mononuclear cells after the family's consent. Erythroblasts produced from these iPSCs exhibited decreased VPS4A localization at the spindle poles and midbody and fail to divide properly, frequently maintaining cytoplasmic bridges as seen in the patient's bone marrow. Additionally, flow cytometry analysis of the patient's peripheral blood cells stained with anti-CD71 for the transferrin receptor and Thiazol Orange (TO) for RNA revealed a unique cell population which is TO negative, yet CD71 positive implying that VPS4A is also involved in reticulocyte maturation, likely participating in vesicle formation and the normal exocytosis of the transferrin receptor.VPS4A appears to play a critical role in erythroblast mitosis and cytokinesis, as well as erythrocyte maturation, and is a novel candidate gene for congenital dyserythropoietic anemia.Figure 1. A) Binucleated erythroblasts and cytoplasmic bridges (arrows) were noted on the patient's bone marrow aspirate smears. B) VPS4A localizes at the spindle poles (upper image) and midbody (lower image) in normal human erythroblasts. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Label-free fluorescent sensor based on aptamer and thiazole orange for the detection of tetracycline

A label-free fluorescence aptasensor based on tetracycline-binding aptamers and thiazole orange (TO) was established for the selective and sensitive detection of tetracycline. TO is essentially nonfluorescent in aqueous solution, while TO can intercalate into the tetracycline-binding aptamers with G-quadruplex structures causing a high fluorescence emission intensity. In the presence of the target tetracycline, the specific recognition and binding of aptamers with the targets induce the conformational changes of aptamers from G-quadruplex structures to hairpin structures. Such target-induced conformational changes lead to the quenching of the fluorescence emission from TO, which thus constitutes the mechanism of the aptamer-based label-free fluorescence biosensor for specific target analysis. Under the optimum conditions, the proposed fluorescence assay shows a linear range from 0.05 to 100 μg mL−1 with a detection limit of 0.029 μg mL−1. The cross-reactivity of this approach against other common antibiotics was negligible compared with the response to tetracycline, proving the excellent selectivity of this label-free fluorescent aptasensor. When used in samples of tetracycline-spiked raw milk, this method demonstrated the satisfactory recoveries between 90.0% and 108.0%, and the results were in full agreement with those from HPLC, confirming the high reliability and feasibility of this bioassay for rapid screening of tetracycline in various practical applications. Using the structure-switching aptamers and the fluorescent intercalator of nucleic acids, the proposed label-free strategy avoids all complicated covalent modifications or chemical labeling, and thus offers obvious advantages of simplicity, convenience, specificity and cost efficiency.

Adaptations in the physiological heterogeneity and viability of Shigella dysenteriae, Shigella flexneri and Salmonella typhimurium, after exposure to simulated gastric acid fluid

Stomach acidity is an important barrier of the human body to protect itself from microbial pathogens entering the small intestine and causing infection. This study examined the survival adaptations of non-acid adapted diarrheal Shigella and Salmonella strains in an environment mimicking the human stomach. The bacterial responses to the challenge of acidic simulated gastric fluid were studied using flow cytometry physiological heterogeneity, membrane integrity and survival (culturability) respectively. Flow cytometry showed that bacterial cells, when exposed to gastric fluid, transformed distinctly, into physiologically heterogeneous sub-populations: intact, stressed and damaged cells, when stained with propidium iodide and thiazole orange. Shigella and Salmonella cells became membrane compromised during initial acid shock (0–30 min), and 80% of these cells shifted to the stressed state throughout gastric fluid exposure. Approximately 10–30% of bacterial strains remained culturable after 60 min of gastric fluid exposure at pH 2.5–4.5, with the percentage increasing with an inoculum size of 102 CFU/ml. This ability of non-acid adapted Shigella and Salmonella sp. to adapt and survive low pH gastric fluid, even though the bacterial numbers decreased or changed to a stressed state, further supports the possible risk of infection when consumed.

Modulating Thiazole Orange Aggregation in Giant Lipid Vesicles: Photophysical Study Associated with FLIM and FCS.

Thiazole orange (TO) exists mainly as a monomer in aqueous medium, where its fluorescence is negligibly small due to intramolecular movements. In the present study, it has been shown that in presence of giant unilamellar vesicles, produced from anionic lipid molecules, TO prefers to form H-dimer and H-aggregates at low lipid concentrations. The nonfluorescent form of TO (monomer) starts fluorescing in the aggregated or dimeric forms. At higher 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) concentration, the TO aggregates disintegrate to the monomeric variants. This is principally due to generation of more surface of residence for the TO molecules. The dye molecules/aggregates reside on the outer surface as well as percolate inside the lipid vesicles toward the inner water pool due to the presence of anionic charges at the interface. We adopted fluorescence lifetime imaging to find out the heterogeneity in photophysics of the different forms of TO inside the lipid vesicles supported by fluorescence correlation spectroscopy to characterize the formation or disintegration of the TO aggregates.

Flow cytometric analysis of viable bacteria in urine samples of febrile patients at the emergency department.

Fast and reliable diagnostics are important in febrile patients admitted to the emergency department. Current urine diagnostics are fast but moderately reliable or reliable but time consuming. Flow cytometry (FC) is a new promising technique in the diagnostics of complicated urinary tract infections by counting bacteria in urine samples. The aim of this study is to improve the FC method by counting only viable bacteria. Urine was obtained from 135 consecutive febrile patients at the emergency department. According to protocol regular diagnostic urine tests were performed. In addition, FC counting of viable and non-viable bacteria was executed after staining with thiazole orange and propidium iodide. All test results were compared to the results of urine culture (≥ 105 colony forming units/mL). At a cut-off value of 2.01 × 105 viable bacteria/mL the sensitivity was 100% and specificity was 78.4% (AUC-value 0.955 on ROC-curve). Spearman correlation test exhibited a higher correlation for flow cytometric counting of only viable bacteria than counting of all bacteria (0.59 vs. 0.37). Using ROC-curves, the AUC-values for FC counting of all bacteria, only viable bacteria and Gram staining were respectively 0.935, 0.955, and 0.968 (P > 0.05). FC counting of only viable bacteria can predict quickly and reliably positive and negative urine cultures in febrile patients admitted to the emergency department. It can help to improve the speed and accuracy of the diagnostic procedure at the emergency department. © 2017 Clinical Cytometry Society.

Resuscitation and growth kinetics of sub-lethally injured Listeria monocytogenes strains following fluorescence activated cell sorting (FACS)

This study investigated the effect of acid (pH4.2), osmotic (10% NaCl) and heat (55°C for 30min) stress induced injury on Listeria monocytogenes strains ATCC19115, 69, 159/10 and 243 using differential plating and flow cytometry coupled with membrane integrity indicators, thiazole orange (TO) and propidium iodide (PI) staining. Growth kinetics of injured cells sorted by fluorescence activated cell sorting (FACS) were studied at 4, 25 and 37°C. The percentage of cell injury detectable by both flow cytometry and differential plating varied significantly among strains and stress treatments (p<0.0001). Based on flow cytometry and TO/PI staining, acid stress caused the highest level of injury followed by heat and osmotic stress. Following cell sorting, acid and osmotic stress injured cells were capable of resuscitation and re-growth while heat injured cells (except for strain 69) were incapable of re-growth despite having a high level of membrane intact cells. The lag phase duration (λ) of sorted stress injured cells resuscitated in brain heart infusion (BHI) broth was significantly influenced by strain variations (p<0.0001), stress treatments (p=0.007) and temperature of resuscitation (p≤0.001). Following repair, the maximum specific growth rate (μmax) of resuscitated cells was not different from untreated control cells regardless of strain differences and stress treatments. Only temperature had a significant effect (p<0.0001) on growth rate. Sorted cells were also capable of growth at 4°C, with the time to detectable growth (≥1.40Log10CFUml−1) ranging from 3 to 15days. Overall, re-growth potential of sorted cells showed that while membrane integrity was a good indicator of cell injury and viability loss for acid and osmotic stress, it was not a sufficient indicator of heat stress injury. Once injured cells repair the cellular damage, their growth rate is not different from non-injured cells regardless of form of stress and strain differences. Thus highlighting the potential food safety risks of stress injured L. monocytogenes cells.

Optical determination of the electronic coupling and intercalation geometry of thiazole orange homodimer in DNA.

Sequence-selective bis-intercalating dyes exhibit large increases in fluorescence in the presence of specific DNA sequences. This property makes this class of fluorophore of particular importance to biosensing and super-resolution imaging. Here we report ultrafast transient anisotropy measurements of resonance energy transfer (RET) between thiazole orange (TO) molecules in a complex formed between the homodimer TOTO and double-stranded (ds) DNA. Biexponential homo-RET dynamics suggest two subpopulations within the ensemble: 80% intercalated and 20% non-intercalated. Based on the application of the transition density cube method to describe the electronic coupling and Monte Carlo simulations of the TOTO/dsDNA geometry, the dihedral angle between intercalated TO molecules is estimated to be 81° ± 5°, corresponding to a coupling strength of 45 ± 22 cm-1. Dye intercalation with this geometry is found to occur independently of the underlying DNA sequence, despite the known preference of TOTO for the nucleobase sequence CTAG. The non-intercalated subpopulation is inferred to have a mean inter-dye separation distance of 19 Å, corresponding to coupling strengths between 0 and 25 cm-1. This information is important to enable the rational design of energy transfer systems that utilize TOTO as a relay dye. The approach used here is generally applicable to determining the electronic coupling strength and intercalation configuration of other dimeric bis-intercalators.

Supramolecular Assembly Mediated by Metal Ions in Aqueous Solution and Its Application in Their Analysis.

A water-soluble fluorescent sensor based on the interaction of twisted cucurbit[14]uril (tQ[14]) and thiazole orange (TO) in aqueous solution was developed. Formation of the tQ[14]/TO complex gives rise to stronger fluorescence in both neutral and acidic aqueous solutions compared with that of free TO. Further experiments on the interaction of the tQ[14]/TO complex with metal ions revealed that, from a series of nineteen selected common metal ions, 1) only Hg2+ can lead to fluorescence enhancement and then quenching of the tQ[14]/TO (2:1) complex in neutral aqueous solution; 2) only Ba2+ can induce fluorescence quenching of the tQ[14]/TO (2:1) complex in aqueous HCl solution (pH 2); furthermore, the tQ[14]/TO/Ba2+ systems exhibit reversible changes in fluorescent intensity on successively adding SO42- and Ba2+ ; and 3) only Ba2+ or Pb2+ can induce fluorescence quenching of the tQ[14]/TO (15:1) complex in aqueous HCl solution (pH 2). Thus, the tQ[14]/TO complex can act as a supramolecular fluorescence-based sensor for the determination of Hg2+ , Ba2+ , and Pb2+ ions.

A highly sensitive and widely adaptable plasmonic aptasensor using berberine for small-molecule detection

Localized surface plasmon resonance (LSPR) biosensors allow label-free detection of small molecules in molecular binding events; however, they are limited by a relatively low sensitivity and narrow dynamic range. Here, we report highly sensitive small-molecule detection by LSPR peak shift exploiting the G-quadruplex (GQx) structure-binding characteristic of known GQx binders to enhance the LSPR signal of a plasmonic aptasensor. Six known GQx binders (thiazole orange, malachite green, crystal violet, zinc protoporphyrin IX, thioflavin T, and berberine) were tested for their ability to enhance the LSPR signal. Among these, berberine (BER) induced the largest LSPR peak shift by interacting with the GQx structure formed by the aptamer/target binding event on a gold nanorod surface. This specific binding performance was confirmed by the fluorescence signal of BER and through repeated cycles of BER addition and washing on the plasmonic sensing chip. The proposed plasmonic aptasensor respectively showed limit of detection (LOD) of 0.56, 0.63, 0.87 and 1.05 pM for ochratoxin A, aflatoxin B1, adenosine triphosphate and potassium ions, which was 1000-fold higher than that in BER-free condition, and a wide dynamic range from 10 pM to 10μM. In addition, the proposed LSPR aptasensor could effectively be used to quantitatively analyze small molecules in real samples.

Structural basis for high-affinity fluorophore binding and activation by RNA Mango.

Genetically encoded fluorescent protein tags revolutionized proteome studies, while the lack of intrinsically fluorescent RNAs has hindered transcriptome exploration. Among several RNA-fluorophore complexes that potentially address this problem, RNA Mango has an exceptionally high affinity for its thiazole orange (TO)-derived fluorophore, TO1-Biotin (Kd ~3 nM), and in complex with related ligands, is one of the most red-shifted fluorescent macromolecular tags known. To elucidate how this small aptamer exhibits such properties, which make it well suited for studying low-copy cellular RNAs, we determined its 1.7 Å resolution co-crystal structure. Unexpectedly, the entire ligand, including TO, biotin, and the linker connecting them, abuts one of the near-planar faces of the three-tiered G-quadruplex. The two heterocycles of TO are held in place by two loop adenines and make a 45° angle with respect to each other. Minimizing this angle would increase quantum yield and further improve this tool for in vivo RNA visualization.

A Thiazole Orange Derivative Targeting the Bacterial Protein FtsZ Shows Potent Antibacterial Activity.

The prevalence of multidrug resistance among clinically significant bacteria calls for the urgent development of new antibiotics with novel mechanisms of action. In this study, a new small molecule exhibiting excellent inhibition of bacterial cell division with potent antibacterial activity was discovered through cell-based screening. The compound exhibits a broad spectrum of bactericidal activity, including the methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and NDM-1 Escherichia coli. The in vitro and in vivo results suggested that this compound disrupts the dynamic assembly of FtsZ protein and Z-ring formation through stimulating FtsZ polymerization. Moreover, this compound exhibits no activity on mammalian tubulin polymerization and shows low cytotoxicity on mammalian cells. Taken together, these findings could provide a new chemotype for development of antibacterials with FtsZ as the target.

The interaction of a structural flexible small molecule with nucleic acid structures: Investigation of the origin of fluorescence signal discrimination in sensing and the utilization in live cell imaging

Thiazole orange (TO) stains most nucleic acids giving strong fluorescence signal non-specifically. To resolve the poor discriminating ability of TO among tertiary structures of DNAs, a planar and rotatable π–conjugated small molecule designed by replacing the methylene bridge with an ethylene that slightly increases its structural flexibility, while maintain the crucial conjugated co-planar two-ring system. The study addressed the origin of signal discrimination for the molecule upon binding with different DNA structures and the results demonstrate that the molecule exhibits excellent fluorescence signal discrimination between the DNA G-quadruplexes and other DNA tertiary structures. In a typical staining assay, the molecule induced 30-fold greater fluorescence signal upon binding with Pu27 than that of ssDNA and dsDNA. Molecular modelling studies also support that the flexible ethylene bridge between the two-ring system is the crucial factor for inducing selectivity towards G-quadruplexes. Furthermore, the selectivity, sensitivity, and stability of the molecule were investigated with various techniques. The application of the molecule as a selective staining agent for imaging of G-quadruplex in human prostate cancer cells PC3 and mouse fibroblast L929 was also investigated. The strongly observed green fluorescence signal in the cell nucleus suggests that the molecule is able to enter into the nucleus and then binding with G-quadruplexes.

Rationally designed double emulsion process for co-encapsulation of hybrid cargo in stealth nanocarriers

Double emulsion process has become highly promising for development of PEG-ylated nanocarriers (NCs) with co-encapsulated hybrid model agents, i.e., hydrophilic deoxyribonucleic acid (DNA) and hydrophobic Thiazole Orange (TO) dye, in the double compartment structure to protect them from the environmental conditions and to investigate different parameters affecting the size, charge and morphology as well as colloidal and biological stability of the final theranostic nanosystems. Different stabilizing agents including surfactants: Cremophor A25, Cremophor RH 40, Poloxamer 407, di-C12DMAB as well as polymers: PEG-PDLLA, PEG-PLGA, PEG-PCL, were screened to choose suitable ones for this approach. The average size of the synthesized NCs measured by dynamic light scattering (DLS) remained <200nm. The encapsulation efficiency of the hybrid cargo was confirmed by UV–vis spectroscopy. Morphology and shape of the loaded nanocontainers were investigated by transmission electron microscopy (TEM) and atomic force microscopy (AFM). Time-depended colloidal stability studies with DLS and ζ-potential followed by turbidimetric technique allow to select only the long-term nanosystems to final investigation the “stealth” properties of the fabricated PEGylated NCs.

Young steady-state rabbit platelets do not have an enhanced capacity to expose procoagulant phosphatidylserine

Platelets are recognized to be physiologically and functionally heterogeneous. An example of the diversity in reactivity is the formation of a distinct subpopulation of procoagulant phosphatidylserine (PS)-exposing platelets upon activation. Platelet age has been proposed as a determinant of platelet function, and it has been reported that young platelets are more reactive in exposing PS; using the same methodology of thiazole orange (TO) staining to distinguish young and old platelets, the percentages of procoagulant platelets produced by thrombin plus collagen activation of platelets from healthy controls were examined by flow cytometry. The procoagulant subpopulation formed by TO-positive platelets (with high TO fluorescence), purported to be young reticulated platelets, was observed to be significantly larger than that formed by TO-negative platelets (with low TO fluorescence), purported to be older platelets. However, it was noted that TO fluorescence in the total platelet population was unimodal and increased with platelet size, assessed by forward scatter. This observation raised the concern that TO-positive platelets are not necessarily the youngest platelets in the condition of steady-state platelet production. Thus, to unequivocally determine whether platelet age is a factor in procoagulant platelet formation, a different approach to identify young, steady-state platelets was employed. Rabbits were injected with biotin to label >95% of circulating platelets in vivo; 24 hours post-biotinylation, the non-biotinylated platelets in the circulation, detected flow cytometrically, are the youngest, newly-formed platelets. It was demonstrated that these youngest platelets were not larger in size than older, biotinylated platelets, and that they did not have an enhanced capacity to expose PS.