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Control of fluorescent molecular assemblies is an exciting area of research with large potential for various important applications, such as, fluorescence sensing/probing, cell imaging and monitoring drug-delivery. In the present contribution, we have demonstrated control on the extent of aggregation of a dye-polyelectrolyte assembly using a macrocyclic host molecule, sulfobutylether-β-cyclodextrin (SBE-β-CD). Initially, a cationic molecular rotor based organic dye, Auramine-O (AuO), undergoes aggregation in the presence of an anionic polyelectrolyte, polystyrene sulfonate (PSS), and displays a broad intense new emission band along with large variation in its absorption features and excited-state lifetime. A manipulation of the monomer-aggregate equilibrium of the dye-polyelectrolyte assembly has been achieved by introducing a cyclodextrin based supramolecular host, SBE-β-CD, which leads to relocation of AuO molecules from polyelectrolyte (PSS) to supramolecular host cavity, owing to the formation of a host-guest complex between AuO and SBE-β-CD. A reversible control on this manipulation of monomer-aggregate equilibrium is further achieved by introducing a competitive guest for the host cavity i. e., 1-Adamantanol. Thus, we have demonstrated an interesting control on the dye-polyelectrolyte aggregate assembly using a supramolecular host molecule which open up exciting possibilities to construct responsive materials using a repertoire of various host-specific guest molecules.

Background The rapid diagnosis of extrapulmonary tuberculosis in children remains challenging. The presence of enlarged lymph nodes provides an opportunity to obtain diagnostic material through fine needle aspiration biopsy (FNAB). Mycobacterial culture, traditionally the reference standard, has a slow turnaround time and PCR-based methods are not widely available in developing countries. Direct visualization of mycobacteria on microscopy can be a rapid method to confirm the diagnosis. This study compared three staining methods to visualize mycobacteria. Methods Hundred FNAB specimens from persistently enlarged lymph nodes in children, clinically suspicious for tuberculosis, were evaluated for the presence of mycobacteria by three staining methods: Papanicolaou induced fluorescence (PIF) and Auramine O staining using fluorescence microscopy and Ziehl-Neelsen (ZN) staining using conventional light microscopy. These methods were evaluated against mycobacterial culture. Results PIF positivity was 30%, with 38% and 48% for Auramine O and ZN respectively. The combined ZN/PIF positivity was 56%. The highest diagnostic accuracy (73%) was demonstrated by ZN alone and in combination with PIF, with PIF alone showing the lowest (49%) accuracy. Although the combined test showed the highest sensitivity, it had the lowest specificity, while ZN was significantly more sensitive than both other staining modalities. No statistical difference in specificity was seen among the tests. Conclusion This study suggests that Auramine O staining on previously ZN stained slides does not significantly improve diagnostic accuracy. While currently widely available methods of direct visualization of mycobacteria suffer from low sensitivity, the ZN stain remains a useful diagnostic test, particularly in resource-constrained countries.

Burning incense to worship deities is a popular religious ritual in large parts of Asia, and is a popular custom affecting more than 1.5 billion adherents. Due to incomplete combustion, burning incense has been well recognized to generate airborne hazards to human health. However, the correlation between burning incense and lung cancer in epidemiological studies remains controversy. Therefore, we speculated that some unknown materials in incense smoke are involved in the initiation or progression of lung cancer. Based on this hypothesis, we identified a major compound auramine O (AuO) from the water‐soluble fraction of incense burned condensate using mass spectrometry. AuO is commonly used in incense manufacture as a colorant. Due to thermostable, AuO released from burned incenses becomes an unexpected air pollutant. AuO is classified as a Group 2B chemical by the International Agency of Research on Cancer (IARC), however, the damage of AuO to the respiratory system remains elusive. Our study revealed that AuO has no apparent effect on malignant transformation; but, it dramatically promotes lung cancer malignancy. AuO accumulates in the nucleus and induces the autophagy activity in lung tumor cells. AuO significantly enhances migration and invasive abilities and the in vitro and in vivo stemness features of lung tumor cells through activating the expression of aldehyde dehydrogenase family 1 member A1 (ALDH1A1), and ALDH1A1 knockdown attenuates AuO‐induced autophagy activity and blocks AuO‐induced lung tumor malignancy. In conclusion, we found that AuO, an ingredient of incense smoke, significantly increases the metastatic abilities and stemness characters of lung tumor cells through the activation of ALDH1A1, which is known to be associated with poor outcome and progression of lung cancer. For public health, reducing or avoiding the use of AuO in incense is recommended.

OBJECTIVE: We sought to evaluate the effectiveness of the auramine orange (AO) stain in diagnosing mycobacterial cervical adenitis (MCA) from fine needle aspiration (FNA) cytology. METHODS: A retrospective review of 19 patients evaluated at 2 urban hospitals from 2000 to 2003 for suspected MCA. FNA specimens were inoculated to culture media and had direct smears stained by the auramine acid fast method.RESULTS: Mycobacteria were identified in 16 (84.2%) of 19 AO-stained FNA specimens, with results available within 4 hours. Corresponding cultures were positive for mycobacteria in 12 specimens, 9 tuberculous and 3 nontuberculous, and grew Mycobacterium tuberculosis from the 3 AO-negative specimens. Three of the 4 patients with negative cultures had previously taken anti-mycobacterial medications.CONCLUSION: The AO stain with fluorescence microscopy is a sensitive and rapid method for detecting tuberculous and nontuberculous mycobacteria. It is a valuable tool for the otolaryngologists and pathologists in th...

Flow cytometrical methods have been introduced recently as an alternative to the enumeration of reticulocytes by microscopy. Two of these methods have gained widespread use in haematological practice; the multiparametric flow cytometer using thiazole orange staining (Retic-Count, FACScan) and the single-application reticulocyte counter using auramine-O staining (R-series, Sysmex). Several studies have emphasized the excellent correlations between microscopy and these techniques. The purpose of our study has been to examine the specificity of these automated devices with regard to cells classified as 'reticulocytes' and the effect that this may have on measures of reticulocyte maturity. Our results indicate that the specificity of reticulocyte measurements by both the Sysmex R-1000/-3000 and the Retic-Count system is relatively low. This is due to the presence of leucocytes amongst cells classified as reticulocytes. These leucocytes display intense staining with either dye, leading to an erroneous estimation of RMI (thiazole orange) and high fluorescence count (R-1000/-3000). This error is directly correlated with the leucocyte count. The basis for reticulocyte identification should be improved before automated estimation of reticulocyte maturation can be used in clinical practice.

The interaction of yeast glyceraldehyde 3-phosphate dehydrogenase with a cationic fluorescent dye, auramine O, was studied by equilibrium dialysis, circular dichroism and fluorescence spectroscopy. Two equal and independent dye binding sites per tetrameric enzyme were detected with a dissociation constant of (4.5 ± 1) × 10−5 M. The maximum fluorescence of bound auramine O is at 515 nm, the excitation spectrum is characterized by the maxima at 380 nm and in the region of 460–470 nm. The complex of auramine O with the enzyme exhibits circular dichroism with a maximum at about 455 nm. The binding of NADH has no effect on the enzyme-dye interaction, whereas the oxidized coenzyme and nicotinamide mononucleotide displace auramine O from the protein. Nicotinamide mononucleotide was shown to induce conformational changes upon binding to the enzyme and to be capable of serving as a coenzyme in the dehydrogenase reaction. AMP, ADP and ADP-ribose form ternary complexes with the enzyme-dye binary complex. This results in a marked increase in fluorescence of the bound dye and was used to characterize the interaction between the enzyme and the adenine-containing NAD+ fragments. In the presence of glyceraldehyde 3-phosphate, the fluorescence of the bound auramine O is quenched, the dye-protein association constant remaining unchanged. Glyceraldehyde, a non-phosphorylated substrate analog, and glycerol 3-phosphate, a competitive inhibitor, are without any effect. Modification of the active-site cysteine-149 with iodoacetate does not affect the dye binding or fluorescence, whereas modification with iodoacetamide diminishes the fluorescence of the enzyme-bound dye without altering the dissociation constant. The effect of adenine-containing NAD+ fragments on the fluorescence of the enzyme-bound dye does not change upon modification of cysteine-149. Auramine O, added together with ADP-ribose, protects the apoenzyme against the inactivation due to modification of essential arginine residues. The effect is similar to that of NAD+ and is absent with auramine O alone or ADP-ribose alone.

The basis for heparin's ability to accelerate the conversion of auramine O (AuO) to Michlers ketone [P. Band & A. Lukton (1982) Anal. Biochem.120, 19–24] is here explained in terms of well-known polyelectrolyte phenomena. Acceleration of this acidcatalyzed hydrolysis appears to be due to colocalization of the cationic dye and hydronium ion within the microenvironment of heparin's electrostatic domain. The dependence of reaction rate on polymer, dye, and hydronium ion concentration, ionic strength, and various ratios of these parameters is consistent with what others have observed for polyelectrolyte catalysts. Dextran sulfate, polyvinyl sulfate, and polyanethole sulfonate likewise accelerate this reaction, thus precluding any explanation of the catalysis in terms of a specific catalytic site. A striking aspect about the polyanion–AuO system is the inability of all other natural glycosaminoglycans tested to catalyze this reaction, despite their analogous polyanionic nature. Manning's limiting laws describing counterion condensation in polyelectrolyte solutions provide a simple context within which to interpret these results. Calculation of the structural linear charge density parameter, ξ, based on analytically determined sulfate-to-hexosamine ratios, reveals that heparin is unique among the glycosaminoglycans in possessing an ξ value greater than unity under the conditions described. Thus, heparin's differential ability to catalyze AuO hydrolysis is a reflection of the fact that only heparin is sulfated to an extent great enough to maintain ξ > 1, even when the negative charge of carboxylate groups is neutralized. It is proposed that this distinction may be important to the unique biochemical attributes of heparin and that such considerations may prove useful in establishing structure–function relationships when comparing different glycosaminoglycan classes.

The association of imidazole and auramine O to native horse-liver alcohol dehydrogenase [Zn(II)LADH] and active-site specifically cobalt(II)-substituted horse-liver alcohol dehydrogenase [Co(II)LADH], respectively, has been investigated. In all cases [except imidazole binding to Zn(II)LADH in the presence of auramine O] the association rates approached an upper limit (kmax). The kmax values were compared for the metal ligands imidazole (monodentate), 1,10-phenanthroline and 2,2'-bipyridine (bidentate; see also the preceding paper), and for auramine O which does not coordinate to the catalytic metal ion. Independent of the large differences in their structure and metal-bonding capability, all these compounds exhibit common, maximum, limiting rate constants of about 60 s-1 and 200 s-1 for Co(II)LADH and Zn(II)LADH, respectively. These results demonstrate that kmax is strongly dependent on the catalytic metal ion but not on the ligand. The absence of spectral changes in the d-d transitions of the catalytic Co(II) ion upon auramine O binding to Co(II)LADH indicates that the rate-limiting step is not accompanied by a major conformational change. Finally, it is concluded that reactions in the inner coordination sphere of the catalytic metal ion (i.e. the metal-bound water molecule) are not responsible for the step characterized by kmax. We propose the rate-limiting step to consist of the dissociation of one or several water molecules from the second coordination sphere of the catalytic metal ion in the active site of LADH in its open conformation.