One Stone, Three Birds: pH Triggered Transformation of Aminopyronine and Iminopyronine Based Lysosome Targeting Viscosity Probe for Cancer Visualization.

The lysosomes of cancer cells have lower pH and higher viscosity than those of normal cells. These features can be used as sensitive and selective markers for cancer diagnosis. In this work, a pH and viscosity dual responsive lysosome targeting fluorescent probe 1 was designed based on the transformation of amino- and imino- forms of pyronine and the twisted intramolecular charge shuttle (TICS) sensing mechanism. Live cancer cells and tumors were effectively distinguished from normal cells and organs through fluorescence imaging of probe 1, which indicated that probe 1 could serve as an effective tool for visualization of tumors at organ level with high selectivity.

A Benzopyronin-Based Two-Photon Fluorescent Probe for Ratiometric Imaging of Lysosomal Bisulfite with Complete Spectral Separation.

Bisulfite (HSO3-), which equilibrates with sulfite (SO32-) and sulfur dioxide (SO2) in aqueous media, can be produced endogenously during oxidation of hydrogen sulfide or sulfur-containing amino acids. Lysosomes, known as the scavengers of living cells, play a crucial role in the metabolic process, and bisulfite is often produced inside the lysosomes. Therefore, detection of bisulfite in lysosomes is a subject of significant interest. Herein, we disclose a lysosome-targeting, two-photon excitable, and ratiometric signaling (near-infrared/green) fluorescent probe that detects bisulfite through a fast 1,6-conjugate addition reaction. The probe shows excellent selectivity toward bisulfite over other biologically relevant species. Notably, the probe allows ratiometric fluorescence imaging of lysosomal bisulfite with complete spectral separation under one-photon as well as two-photon excitation conditions.

In situ forming interpenetrating hydrogels of hyaluronic acid hybridized with iron oxide nanoparticles.

Four derivatives of hyaluronic acid (HA) bearing thiol (HA-SH), hydrazide (HA-hy), 2-dithiopyridyl (HA-SSPy), and aldehyde groups (HA-al) respectively were synthesized. Thiol and 2-dithiopyridyl as well as hydrazide and aldehyde make up two chemically orthogonal pairs of chemo-selective functionalities that allow in situ formation of interpenetrating (IPN) disulfide and hydrazone networks simultaneously upon the mixing of the above derivatives at once. The formation of IPN was demonstrated by comparing it with the formulations of the same total HA concentration but lacking one of the reactive components. The hydrogel composed of all four components was characterized by a larger elastic modulus than those of the control single networks (either disulfide or hydrazone) and the three component formulations gave the softest hydrogels. Moreover, a hydrazone cross-linkage was designed to contain a 1,2-diol fragment. This allowed us to partially disassemble one type of network in the IPN leaving another one unaffected. In particular, treatment of the IPN with either sodium periodate or dithiothreitol resulted in disassembly of the hydrazone and disulfide networks respectively and thus softening of the hydrogel. Contrarily, the single network hydrogels completely dissolved under the corresponding conditions. In corroboration with this, enzymatic degradation of the IPN by hyaluronidase was also substantially slower than the degradation of the single networks. In order to further improve the mechanical properties of the elaborated injectable IPN, it has been in situ hybridized with iron oxide nanoparticles (IONPs). The mesh size of the IPN was smaller than the size of the IONPs resulting in the retention of nanoparticles in the matrix under equilibrium swelling conditions. However, these nanoparticles were released upon enzymatic degradation suggesting their use as MRI tags for non-invasive tracking of the hydrogel material in vivo. Additionally, this injectable hybridized hydrogel with encapsulated IONPs can be used in hyperthermia cancer therapy.

Inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment.

The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.

Quick staining technique for myeloperoxidase using potassium iodide and oxidized pyronine B.

Myeloperoxidase (MPO) staining has been important for the cytomorphological diagnosis and classification of leukemia. A novel staining method for MPO and its clinical application are presented in the report. Pyronine B (PyB), serving as a chromogenic reagent, was pre-oxidized to obtain stable oxidized Pyronine B solution. The MPO working solution for oxidized pyronine B method consisted of phosphate buffer solution, potassium iodide (KI) solution, and oxidized Pyronine B solution. The positive products of the oxidized Pyronine B method of MPO staining were vibrant red particles located in cytoplasm and the nucleus was stained bluish green. Bone marrow smears from 229 patients with acute leukemia or with grossly normal bone marrow were stained by both oxidized Pyronine B method and the conventional Washburn benzidine staining and a comparison revealed no significant difference in the positive detection rate between the two techniques. The new method eliminates the influence of the varying amount of H2O2 on MPO staining. With this method, the reagents were more stable and the staining procedure was simple and time-saving. This MPO staining technique is a better alternative than the conventional benzidine-based methods.

Electroporation of cell membranes supporting penetration of photodynamic active macromolecular chromophore dextrans.

The aim is to demonstrate that macromolecular chromophore dextrans (Cibacron-dextran) acting as photosensitizers can be transported easily into cancer cells by electroporation of their membranes (short electric pulses on cell suspension between electrodes). There are two possibilities, either:(A)irradiation starts with the electropulse-showed with easily penetrating thiopyronin-yielding nearly 100% dead cells;(B)irradiation starts after a resealing time of membrane pores during which macromolecular photosensitizers can penetrate into cells. In this way, fractions of Cibacron-dextran with molecular weights (Mw) 3300, 10,900 and 500,000 are now able to kill. This combination of bioelectrochemistry and photobiology will be suitable also for other biopolymers, connected with photodynamic active chromophores (e.g. chromopeptides) to transport them through cell walls and membranes into cells and tissues. The human cancer cells U-935 and K-562 (pulsed by 1.15 kV/cm field strength) additionally or synergistically reach high rates of necrotic cells (colored by trypan blue) by this combination.

A sensitive double immunostaining protocol for Fos-immunoreactive neurons.

Immunostaining of Fos, the nuclear protein encoded by the immediate early gene c-fos, is widely used to reveal the functional activation of neurons. The chemical identity of cells that express c-fos can be investigated with double immunohistochemistry. We report the usefulness of a sequential two-color avidin-biotin-immunoperoxidase method that provides a highly sensitive double immunostaining and allows long-term storage of the sections. In this protocol, metal intensification of diaminobenzidine (int-DAB) resulted in dark brown/black Fos immunostaining of the neuronal nucleus. The use of alpha-naphthol/pyronin reaction product yielded pink immunostaining of a second antigen in the cytoplasm. This combination produced higher contrast than that produced by int-DAB Fos immunostaining combined with conventional DAB light brown cytoplasmic staining. The sensitivity of the use of int-DAB and alpha-naphthol/pyronin was verified in different experimental paradigms, combining the immunocytochemical detection of Fos with that of the p75 nerve growth factor receptor, or parvalbumin, or calbindin D28k.

Investigation of some antimicrobial procedures on the in vitro development of early murine embryos aimed toward developing methods for the disinfection of mammalian embryos prior to transfer.

Eight-cell, zona pellucida-intact mouse embryos were exposed to the following substances or procedures that have been reported to have germicidal effects to determine if the embryos would survive and develop under in vitro conditions: the photosensitive substances hematoporphyrin, hematoporphyrin derivative, 8-methoxypsoralen, 4,5',8-trimethylpsoralen, and thiopyronine; the enzymes lipase (0.5%), phospholipase C (2 U/ml), chymotrypsin (0.5%), and trypsin (0.5%); pH 5.0; and helium/neon laser light, visible light, ultraviolet A light, and ultraviolet C light. Under the conditions used, embryos were not adversely affected by hematoporphyrin and/or helium/neon laser light; methoxypsoralen and/or ultraviolet A light; lipase; trypsin; pH 5.0 for 20 min; and visible light. Variable results were obtained from hematoporphyrin derivative with laser light. Thiopyronine, trimethylpsoralen in combination with ultraviolet A light, and ultraviolet C light killed embryos, and chymotrypsin and phospholipase C were harmful at 10- and 15-min exposure times, respectively.

UV-induced reactivation and mutagenesis of lambda-phages after treatment with 8-methoxypsoralen or thiopyronine and light.

Lesions, which were produced on lambda-phages DNA by the photosensitization effect of 8-methoxypsoralen (8-MOP) can be repaired by UV-induced repair systems (W-reactivation) in Escherichia coli wild type host cells. By optimum induction of the repair system, about 45% of the 8-MOP lesions are repaired. The survival of lambda-phages inactivated by the photodynamic action of thiopyronine (TP) is only negligibly increased by the same UV-induced repair systems. However, the frequencies of clear plaque mutations of 8-MOP treated as well as TP treated lambda-phages increase in similar fashion if the host cells of wild type have been irradiated with UV. These results show the different capacities of induced repair systems in repairing different types of lesions. They also suggest that some types of base damages are repaired more error-prone than other DNA-lesions.

A rapid method for measuring drug enrichment in epidermis.

A rapid and simple method is described for measuring the enrichment of small molecules in epidermal tissue. To measure such an enrichment, a small tissue sample (2-10 mg) is allowed to equilibrate with a buffered solution of a labelled substance for periods of 12-36 h. The concentration of the radioactive molecule in the tissue is measured as a decrease of radioactivity in the solution. Concentration measurements in the tissue itself can be performed, but are not required to detect enrichment in the tissue or to assess its magnitude. The specific density of appendage free human epidermis has been determined and was found to be 1.20 g/cm3. Using this value, tissue weight can be translated into volume and concentraton changes in the solution can be recalculated to yield the concentration of the substance in the tissue itself. Close agreement was found between the calculated tissue concentration and the values actually measured, following digestion of the epidermis with NaOH and measuring the activity in the tissue digest. The enrichment of five substances in human epidermis was measured: alpha-estradiol, thiopyronincce, 8-methoxypsoralen (8-MOP), 5-methoxypsoralen (5-MOP), and theophylline. Of these substances, the first four are concentrated by human epidermis and the concentrations reached within the tissue are 10-500 times higher than the concentration of the same substance in the surrounding buffer. The enrichment data has been analysed in an attempt to distinguish between reversible affinity binding to specific tissue sites and partitioning of the substances between buffer and tissue components (lipids, membranes, etc.). In the case of thiopyronin and 8-MOP, reversible binding is indicated with dissociation constants of 10(-7) M and 10(-5) M, respectively, while partitioning distribution could account for the behavior of 5-MOP and alpha-estradiol. The method can be used either as a rapid screening method or as a quantitative analysis for the characterization of tissue enrichment with specific drugs.